1. Kunzwisisa kwekutanga
Panguva ino, isu tinofanirwa kunzwisisa mamwe mazwi uye mazwi, kuitira kuti tidzivise kukanganisa pamberi pevakuru vedu, senge:
Mubvunzo: Ndeupi musiyano uripo pakati peRT-PCR, qPCR, Real-time PCR, uye chaiyo-nguva RT-PCR?
Mhinduro: RT-PCR ndeye reverse transcription PCR(reverse transcription PCR, RT-PCR), inova musiyano unoshandiswa zvakanyanya wepolymerase chain reaction (PCR).MuRT-PCR, RNA strand inodzoserwa kumashure kuita DNA inopindirana, iyo inobva yashandiswa setemplate yekukwidziridzwa kweDNA nePCR.
Chaiyo-nguva-PCR uye qPCR(Quantitative Rea-ltime-PCR) chinhu chimwe chete, zvose ndezvechokwadi-nguva quantitative PCR, izvo zvinoreva kuti imwe neimwe yePCR ine zvinyorwa zve data chaiyo, saka nhamba yekutanga templates inogona kugadziriswa kunyatsoongorora.
Kunyangwe zvese zviri zviviri Real-time PCR (chaiyo-nguva fluorescent quantitative PCR) uye Reverse transcript PCR (reverse transcription PCR) ichiita seyakapfupikiswa seRT-PCR, gungano repasi rese nderokuti: RT-PCR inonyatso reva kudzoreredza kunyorwa.PCR, Real-time PCR inowanzopfupikiswa se qPCR (huwandu chaihwo-nguva PCR).
Uye chaiyo-nguva RT-PCR (RT-qPCR), Ndiyo reverse transcription PCR yakasanganiswa nefluorescent quantitative tekinoroji.: tanga wawana cDNA (RT) kubva kuRNA reverse transcription, wobva washandisa Real-time PCR yehuwandu hwekuongorora (qPCR).Marabhoritari mazhinji anoita RT-qPCR, ndiko kuti, tsvakiridzo paRNA kutaura pasi-mutemo, saka qPCR iyo munhu wese anotaura nezvayo murabhoritari inotoreva RT-qPCR, asi usakanganwa kuti kuchine yakawanda bvunzo dzeDNA mumakiriniki maapplication.Kuongororwa kwehuwandu, sekuonekwa kwehutachiona hweH hepatitis B HBV.
Mubvunzo: Mushure mekuverenga yakawanda fluorescent quantitative PCR, nei chidimbu chakasimudzirwa chichifanira kudzorwa mukati me80-300bp?
Pindura: Kureba kwejini yega yega kutevedzana kwakasiyana, mamwe akati wandei kb, mamwe mazana ebp, asi isu tinongoda chete kuti urefu hwechigadzirwa huve 80-300bp pakugadzira maprimers, mapfupi kana akareba kwazvo haana kukodzera fluorescent quantitative PCR.Chimedu chechigadzirwa chakanyanya kupfupika kuti chisiyaniswe kubva kune primer-dimer.Kureba kweprimer-dimer inenge 30-40bp, uye zvakaoma kusiyanisa kana iri primer-dimer kana chigadzirwa kana iri pasi pe80bp.Kana chidimbu chechigadzirwa chakareba, chinodarika 300bp, chinozotungamira kune yakaderera amplification kunyatsoita uye haigone kunyatsoona huwandu hwejini.
Semuenzaniso, paunoverenga kuti mukirasi mune vanhu vangani, unongoda kuverenga kuti miromo yakawanda sei.Izvi zvakafanana kana iwe uchinge waona majini, iwe unongoda kuona imwe kutevedzana kwejini kumiririra Iyo yese sequence ichaita.Kana uchida kuverenga vanhu, unofanira kuverenga zvose miromo nemhino, nzeve, uye magirazi, uye zviri nyore kukanganisa.
Kuwedzera, mukutsvagisa zvipenyu, kune dzakawanda nyaya dzekutsvagisa kubva pane imwe nzvimbo kuenda kune imwe nzvimbo, nekuti kutevedzana kwemajini echero marudzi akareba kwazvo, hazvina kufanira uye hazvigoneke kuyera zvimedu zvese, senge bhakitiriya 16S kutevedzana, ndiko kuita kutevedzana kwebhakitiriya Assays kuti ape huwandu hwehuwandu hwehutachiona.
Mubvunzo: Ndeupi hurefu hwakakwana hwe qPCR primer dhizaini?
Pindura: Kazhinji kutaura, iyo primer kureba ndeye 20-24bp, izvo zviri nani.Ehe, isu tinofanirwa kutarisisa kukosha kweTM kweiyo primer pakugadzira iyo yekutanga, nekuti izvi zvine hukama neyakakwana annealing tembiricha.Mushure mekuedza kwakawanda, zvakaratidzwa kuti 60 ° C iri nani TM kukosha.Kana iyo annealing tembiricha yakadzikira, inotungamira kune isiri-chaiyo amplification.Kana iyo annealing tembiricha yakawandisa, kugona kwekusimudzira kuchave kwakadzikira, iyo yepamusoro yeamplification curve ichatanga gare gare, uye kukosha kweCT kuchanonoka.
Mubvunzo: Nzira yedhayi yakasiyana sei neye probe nzira?
Mhinduro: Dye nziraMamwe madhayi efluorescent, akadai seSYBR Green Ⅰ, PicoGreen, BEBO, zvichingodaro, haabudise mwenje ari ega, asi anoburitsa fluorescence mushure mekusunga kune diki groove yeDNA ine tambo mbiri.Naizvozvo, pakutanga kwePCR reaction, muchina haugone kuona chiratidzo chefluorescent.Kana kuita kwasvika padanho rekuwedzera-yekuwedzera, tambo yakapetwa kaviri inovhurwa, uye tambo nyowani inogadzirwa pasi pekuita kweDNA polymerase, uye iyo fluorescent molecule inosunga kune dsDNA diki groove.Sezvo huwandu hwePCR cycles huchiwedzera, dhayi dzakawanda uye dzakawanda dzinosanganiswa nekaviri-stranded DNA, uye chiratidzo chefluorescent chinoramba chichiwedzerwa.Nzira yedhayi inonyanya kushandiswa mukutsvakurudza kwesayenzi.
PS: Chenjerera paunenge uchiedza, dhayi inofanira kusanganiswa neDNA yevanhu, chenjerera kuishandura kuva munhu wefluorescent.
Nzira yedhayi (kuruboshwe) Probe nzira (kurudyi)
PS: Chenjerera paunenge uchiedza, dhayi inofanira kusanganiswa neDNA yevanhu, chenjerera kuishandura kuva munhu wefluorescent.
SYBR Green Ⅰ inosungira kune diki groove reDNA
Probe nziraTaqman probe ndiyo inonyanya kushandiswa hydrolysis probe.Kune boka refluorescent pa5 ′ kumagumo kweprobe, kazhinji FAM, uye probe pachayo inoteedzana inoenderana neinotarirwa gene.Pane fluorescent quenching boka pa3 ′ kumagumo.Zvinoenderana nemusimboti wefluorescence resonance simba rekufambisa (Förster resonance simba rekufambisa, FRET), apo mutori wenhau fluorescent boka (donor fluorescent molecule) uye quenching fluorescent boka (inogashira fluorescent molecule) inofara Kana iyo spectra inopindirana uye chinhambwe chiri padhuze (7-10 kuburitsa mamorekuru) , nepo autofluorescence isisina simba.Naizvozvo, pakutanga kwePCR reaction, kana probe yakasununguka uye yakasimba muhurongwa, mutori wenhau fluorescent boka harizobudisi fluorescence.Paunenge uchivhara, primer uye probe inosunga kune template.Munguva yekuwedzera nhanho, iyo polymerase inoramba ichigadzira maketani matsva.DNA polymerase ine 5'-3 ′ exonuclease chiitiko.Kana yasvika pakuferefeta, iyo DNA polymerase inoburitsa hydrolyze kuferefeta kubva patemplate, kupatsanura mutori wenhau fluorescent boka kubva kuquencher fluorescent boka, uye kusunungura chiratidzo chefluorescent.Sezvo pane hukama humwechete-kune-umwe pakati peprobe uye template, nzira yekuongorora ndiyo yepamusoro kune nzira yedhayi maererano nekururama uye kunzwisisika kwekuedzwa.Iyo probe nzira inonyanya kushandiswa pakuongorora.
Q: Chii chinonzi absolute quantification?Chii chinonzi Relative Quantification?
Pindura: Absolute quantification inoreva kuverengwa kwenhamba yekopi yekutanga yesampuli ichaongororwa neqPCR, sekuti mavhairasi mangani eHBV ari mu1ml yeropa.Mhedzisiro yakawanikwa nehuwandu hwehuwandu ndiyo shanduko yehuwandu hweiyo inotarirwa gene mune chaiyo sampu inoenderana neimwe sampu yereferensi, uye gene expression iri kumusoro-yakadzorwa kana pasi-yakadzorwa.
Mubvunzo: Ko huwandu hweRNA kudhirowa, kudzoreredza kunyorwa kwehunyanzvi, uye kukwidziridza kushanda zvakanaka kuchakanganisa mhinduro dzekuyedza?
Mubvunzo: Ko chengetedzo yemuenzanisiro, mareagents ekubvisa, reverse transcription reagents, uye mwenje-kutumira zvinoshandiswa zvinokanganisa mhedzisiro yekuyedza?
Q: Ndeipi nzira inogona kugadzirisa data rekuyedza?
Nezve nyaya idzi, isu tichadzitsanangura zvakadzama muzvikamu zvepamberi uye zvepamusoro pazasi.
2. Ruzivo rwepamusoro
Nezve chaiyo-nguva fluorescent quantitative PCR, tinofanira kuziva chokwadi chekuti zviuru zvemapepa ekutsvagisa kwesainzi anoburitswa gore rega rega, pakati peiyo fluorescent yehuwandu PCR tekinoroji haisi nhamba diki.
Kana pasina chiyero chakajairika chekuyera fluorescent quantitative PCR kuyedza, mhedzisiro inogona kusiyana zvakanyanya.Kune jini rimwechete remhando imwe chete, ine nzira imwechete yekugadzirisa, mibairo yekuona inosiyanawo zvakanyanya, uye zvichanetsa kune vanononoka kudzokorora mhedzisiro imwe chete.Iwe Hapana anoziva chakanaka nechakaipa.
Izvi zvinoreva here kuti fluorescent quantitative PCR inyanzvi yekubiridzira kana tekinoroji isingavimbike?Kwete, imhaka yekuti fluorescent quantitative PCR inonyanya kunzwisiswa uye yakanyatsojeka, uye zvishoma zvisirizvo oparesheni inoburitsa zvakapesana zvachose.Kurasikirwa kudiki kune chiuru chemamaira kure.Munyori wechinyorwa anogona kuramba achitambudzwa nevanoongorora.Panguva imwecheteyo, vaongorori vejenari vakaomawo kusarudza kubva pane zvakasiyana-siyana zvekuedza.
Zvese mune zvese, zvichiratidza kushaikwa kwekubvumirana mune chaiyo-nguva PCR kuyedza.Kuti izvi zviitike, masayendisiti epamusoro muindasitiri akatanga kugadzira zviyero,inoda kuti vanopa rubatsiro vape mamwe anodiwa ekuyedza uye kugadzirisa data (kusanganisira inodiwa data) muchinyorwa kuti isangane nezviyero izvi.
Vaongorori vanogona kutonga kunaka kwekuyedza nekuverenga izvi zvakadzama;vaverengi venguva yemberi vanogona zvakare kushandisa izvi kudzokorora chiedzo kana kuvandudza chiedzo.Ipapo mibairo yekuedzwa inowanikwa nenzira iyi yakazara neruzivo, mhando yepamusoro, uye inoshandiswa.
MIBBI (Ruzivo Rushoma rweBiological uye Biomedical Investigations -http://www.mibbi.org) zvakavapo.MIBBI ipurojekiti inopa zviyero zvezviedzo.Inobudiswa muzvakasikwa.Iyi purojekiti yakanangana neakasiyana ongororo yebhayoloji, kusanganisira cell biology, Microarray, qPCR yatiri kuzokurukura izvozvi, nezvimwewo, uye inopa mhando yega yega yekuyedza paunenge uchiendesa zvinyorwa.Mashoko iwayo anofanira kupiwa nguva dzose.
Muchirongwa cheMIBBI, pane zvinyorwa zviviri zvine chekuita nefluorescent quantitative PCR, zvinoti.:
·RDML (Real-Time PCR Data Markup Mutauro) - mutauro wakarongeka uye gwara rekuzivisa kune chaiyo-nguva yakawanda PCR data;
·MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) - ruzivo rudiki rwekuburitsa zvinyorwa nezve-chaiyo-nguva uwandu PCR zviedzo.
Kutanga, ngatitaure nezve RDML, iyo terminology yakatarwa.
Kana pasina tsananguro yakajairika yezvese, hazvibviri kuenderera mberi nehurukuro, ndosaka tsananguro yematemu yakakosha mubvunzo.
Izwi rinoshandiswa mufluorescent quantitative PCR kuyedza rinosanganisira zvinotevera zvirimo.QIAGEN akatiitira pfupiso yakanakisa.Izvi zvinotevera zvose zvakaomazvinhu .
Amplification curve
Iyo amplification curve inoreva iyo curve yakagadzirwa panguva yePCR maitiro, ine nhamba yekutenderera seabscissa uye chaiyo-nguva fluorescence kusimba panguva yekuita seyakarongeka.
Yakanakisa amplification curve inofanirwa kuve neanotevera maitiro: iyo yekutanga yakadzika kana yakadzikira zvishoma, uye hapana chiri pachena chinokwira kumusoro;iyo inflection point yecurve yakajeka, uye kutsetseka kweexponential phase kunoenderana neamplification kunyatsoshanda.Kuwanda kwemateru, ndiko kuwedzera kwesimba rekusimudzira;iyo yakazara amplification curve Iyo parallelism yakanaka, zvichiratidza kuti iyo amplification inobudirira yechubhu imwe neimwe yakafanana;iyo exponential phase yeamplification curve yeakaderera-concentration samples iri pachena.
Baseline (Baseline)
Iyo yekutanga ndiyo nhanho yeruzha rwekutanga kutenderera, kazhinji kuyerwa pakati pe 3rd uye 15th cycle, nokuti fluorescence kukosha kuwedzera kunokonzerwa neamplification chigadzirwa haigoni kuonekwa panguva ino.Huwandu hwemadenderedzwa anoshandiswa kuverenga hwaro hunogona kusiana uye hungada kudzikiswa kana huwandu hwematemplate huchishandiswa kana nhanho yekutaura yejini inotangwa yakakwira.
Kuisa iyo yekutanga kunoda kutarisa fluorescence data kubva kumutsara amplification curve.Iyo yekutanga inoiswa kuitira kuti kukura kweiyo amplification curve kunotanga nenhamba yekutenderera yakakura kupfuura yekutanga kutenderera nhamba yepamusoro.Baseline inofanirwa kusetwa yega yega kune yega yega chinangwa kutevedzana.Avhareji fluorescence values akaonekwa mukutanga kutenderera anofanirwa kubviswa kubva kune fluorescence values inowanikwa mune yakawedzera zvigadzirwa.Iwo achangoburwa vhezheni akasiyana-siyana Real-Nguva PCR software inobvumira otomatiki optimization yemasetirwo ekutanga emasampuli ega.
Munguva yekutanga mashoma ekutenderera kwePCR amplification reaction, iyo fluorescence chiratidzo hachinje zvakanyanya.Kusvika pamutsara wakatwasuka kunonzi danho rekutanga, asi kana tikanyatso tarisisa pamatanho mashoma ekutanga, tinoona kuti mukati meiyo yekutanga pane zviri kuitika mumufananidzo uri pazasi.
Background Background inoreva
iyo isiri-specific fluorescence kukosha mukuita.Somuenzaniso: kusashanda fluorescence quenching;kana nhamba huru yekaviri-yakasungwa DNA templates nekuda kwekushandiswa kweSYBR Green.Izvo zvikamu zvekumashure zvechiratidzo zvinobviswa nemasvomhu neiyo Real-Time PCR software algorithm.
Mutori wenhau chiratidzo
Mutori wenhau anoreva chiratidzo chefluorescent chakagadzirwa neSYBR Green kana fluorescently yakanyorwa kutevedzana-chaiyo probes panguva Yechokwadi-Nguva PCR.
Normalized Reporter Signal (RN)
RN inoreva kusimba kwefluorescence yemutori wenhau yakakamurwa neiyo fluorescence kusimba kweiyo passive referensi dhayi inoyerwa pakutenderera kwega kwega.
Passive Reference Dye
Mune mamwe maPCR chaiwo,iyo fluorescent dhayi ROX inoshandiswa sereferensi yemukati kugadzirisa chiratidzo chefluorescent.Inogadzirisa misiyano nekuda kwepipetting isina kururama, tsime chinzvimbo, uye fluorescence kuchinjika pane zvakanaka-ne-tsime.
Iyo fluorescence chikumbaridzo (chikumbaridzo)
yakagadziridzwa pamusoro pekukosha kwemashure uye zvakanyanya pasi peiyo plateau kukosha kweiyo amplification curve.Inofanirwa kurara munharaunda yemutsara weiyo amplification curve, inomiririra iyo log-linear renji yekuonekwa kwePCR.Ma Threshold anofanirwa kuiswa muiyo log-amplification curve view kuitira kuti log-linear chikamu chePCR chionekwe zviri nyore.Kana paine akawanda anonangwa majini muReal-Time PCR, chikumbaridzo chinofanirwa kusetwa kune chimwe nechimwe chinangwa .Kazhinji, chiratidzo chefluorescence chekutanga 15 kutenderera kwePCR reaction chinoshandiswa seye fluorescence yekumashure chiratidzo, uye fluorescence chikumbaridzo ndeye 10 nguva yakajairwa kutsauka kweiyo fluorescence chiratidzo chekutanga 3 kusvika ku15 kutenderera kwePCR, uye fluorescence chikumbaridzo chakaiswa muPCR exponplential.Kazhinji, chiridzwa chega chega chine fluorescence pachikumbaridzo chakaiswa chisati chashandiswa.
Cycle Threshold (CT) kana Crossing Point (CP)
Kutenderera uko iyo amplification curve inoyambuka pachikumbaridzo (kureva, iyo nzvimbo iyo fluorescence kuonekwa inowedzera zvakanyanya).CT inogona kuva chidimbu uye huwandu hwekutanga template hunogona kuverengwa.Iko CT kukosha inomiririra huwandu hwema cycles anowanikwa apo chiratidzo chefluorescent mune imwe neimwe PCR reaction tube inosvika pachikumbaridzo chakaiswa.Pane hukama hwemutsara pakati pekukosha kweCT yetemplate yega yega uye logarithm yekutanga kopi nhamba yetemplate, iyoyakakwira yekutanga kopi nhamba, iyo diki kukosha kweCT, uye zvinopesana.Mutengo wepamusoro unogona kuitwa nekushandisa chiyero chine nhamba inozivikanwa yekopi yekutanga, umo abscissa inomiririra kukosha kweCT, uye iyo inomiririra inomiririra logarithm yekutanga kopi nhamba.Nokudaro, chero bedzi iyo CT inokosha yemuenzaniso usingazivikanwi inowanikwa, nhamba yekutanga yekopi yemuenzaniso inogona kuverengwa kubva kune yakajairwa curve.
ΔCT kukosha
ΔCT kukosha kunotsananguramutsauko uripo pakati pejini rinotarirwa uye rinoenderana nereferensi rejeni CT kukosha, senge geni rekuchengetedza imba, uye rinoshandiswa kugadzirisa huwandu hwetemplate inoshandiswa:
⇒CT = CT (chinangwa chejeni) - CT (endogenous reference gene)
ΔCT Kukosha
Iyo ΔΔCT kukosha inotsanangura musiyano pakati pezvinoreva ΔΔCT kukosha kwemuenzaniso wekufarira (semuenzaniso, masero anokurudzirwa) uye zvinoreva ΔΔCT kukosha kwemuenzaniso wekutsvaga (semuenzaniso, masero asina kukurudzirwa).Sampuli yereferenzi inonziwo calibration sampuli uye mamwe ese masampuli akajairwa kune aya kuti averenge huwandu:
⇒ΔCT = avhareji ΔCT (muenzaniso wekufarira) - avhareji ΔCT (reference sample)
Endogenous reference genes (endogenous reference genes)
Matanho ekutaura endogenous reference genes, senge ekuchengetedza imba (majini ekuchengetedza imba), haana kusiyana pakati pemasampuli.Kuenzanisa maitiro eC CT yejini rinotaridzirwa kune jini rinotarirwa rinobvumira mutsara wekutaura wejini rinotarirwa kuti rive rakakwana kusvika kuhuwandu hwekupinza RNA kana cDNA (ona chikamu che ΔCT tsika dziri pamusoro).
Internal referensi majini akaruramazvinogoneka kushatiswa kweRNA kana kuvepo kwema enzyme inhibitors muRNA samples, pamwe nekusiyana mune zveRNA zvemukati, reverse transcription performance, nucleic acid recovery, uye sample kubata.Kuti tisarudze iyo yakakwana referensi jena(s), isu takagadzirisa algorithm kuti ibvumire kusarudzwa kwayo kweiyo yakakwana referensi zvinoenderana nekuyedza marongero.
Kudzora mukati
Kutevedzana kwekudzora kunokwidziridzwa mukuita kwakafanana sekutevedzana kwechinangwa uye kuferefetwa neimwe probe yakasiyana (kureva, kuita duplex PCR).Zvidzoreso zvemukati zvinowanzo shandiswa kutonga kunze kwakakundikana amplifications, senge kana tarisiro yekutevedzana isingaonekwe.
Calibration Muenzaniso
Sample yereferensi (semuenzaniso, yakacheneswa RNA kubva musero mutsara kana matishu) inoshandiswa muhuwandu hwekuenzanisa kuenzanisa mamwe ese masampuli kuti aone hukama hwekutaura kwejini.Sampuli yekuenzanisa inogona kuve chero sampuli, asi kazhinji inodzora (semuenzaniso, sampu isina kubatwa kana sampuli kubva panguva zero yekuyedza).
Positive controls
shandisa control reactions nehuwandu hunozivikanwa hwetemplate.Positive controls inowanzo shandiswa kutarisa kuti primer set kana primer-probe set iri kushanda nemazvo uye kuti maitirwo acho akaiswa nemazvo.
Hapana Template Control (NTC)
Maitiro ekudzora ane ese anodiwa zvikamu zveamplification reaction kunze kweiyo template, iyo inowanzotsiviwa nemvura.Iko kushandiswa kweNTC kunogona kuwana kusvibiswa kunokonzerwa neiyo reagent kusvibiswa kana yekunze DNA, nekudaro kuve nechokwadi uye kuvimbika kwedata rekuona.Kukwidziridzwa kwekutonga kweNTC kunoratidza kusvibiswa.
Hapana RT control (NRT)
RNA yekubvisa maitiro inogona kunge iine yasara genomic DNA, inokuvadza zvakanyanya uye ndiyo honzeri inokanganisa mhando yedata uye muvengi chaiwo weqPCR, saka kana uchigadzira zviedzo, inofanirwa kugadzirwa kuti iwedzere kutariswa kweRNA.Pane nzira mbiri, imwe ndeyekugadzira maprimers mhiri kweintrons, imwe ndeyekubvisa zvachose DNA, iyo iri nani, iyo ichakurukurwa gare gare.Iyo NTR control igirazi remashiripiti kuona kusvibiswa kweDNA.Kana paine amplification, zvinoreva kuti pane kusvibiswa.
Mitemo
Miyero ndiyo sampuli dzinozivikanwa kana nhamba dzekopi dzinoshandiswa kugadzira curve yakajairwa.Kuti uve nechokwadi chekugadzikana kwechiyero, iyo gene fragment inowanzoitwa muplasmid uye inoshandiswa seyero.
The standard curve
anowanzo kucheneswa kuita kanenge 5 concentration gradients neyakajairwa chigadzirwa zvinoenderana nereshiyo yakapetwa kaviri, uye mapoinzi mashanu anodhirowewa mumakongisheni eCT value uye nhamba yekukopa, uye mapoinzi akabatana kuita mutsara kugadzira curve yakajairwa.Kune yega yega curve, kutendeseka kwayo kunoda kuongororwa.Kutsetseka ukoshi kunowira pakati -3.3 uye -3.8, uye imwe neimwe yekuisa inoitwa katatu.Pfungwa dzakasiyana zvikuru nedzimwe pfungwa dzinofanira kuraswa.Iko CT kukosha kwemuenzaniso kuti iedzwe inounzwa muyero curve, uye chiyero chekutaura chemuenzaniso wekuedzwa unogona kuverengwa.
Iko CT kukosha kwemuenzaniso kuti iedzwe inounzwa muyero yakajairwa, uye yekutanga kopi nhamba yemuenzaniso wekuedzwa inogona kuverengerwa.
Kubudirira uye Slope
Kutsetseka kweiyo yakajairwa curve inomiririra kugona kweiyo chaiyo-nguva PCR.
·Kutenuka kwe -3.322 kunoratidza kuti PCR amplification inobudirira 1, kana 100% inoshanda, uye huwandu hwePCR chigadzirwa chakapetwa kaviri panguva yega yega.
·Kutenuka kuri pasi pe -3.322 (eg, -3.8) kunoratidza kugona kwePCR
·Mutsetse wakakura kupfuura -3.322 (eg, -3.0) unoratidza kuti PCR inoshanda inoratidzika kunge yakakura kupfuura 100%, izvo zvinoda kuziva, ko kutenderera kumwe chete kwePCR kungagadzira kanopfuura kaviri chigadzirwa chakawedzerwa?Mamiriro ezvinhu aya anoitika muchikamu chisiri-mutsara chePCR reaction, ndiko kuti, kune huwandu hukuru hwekusanyanya kukwidziridzwa.
melting curve
Mushure mekunge kukwidziridzwa kweqPCR kwapera, chigadzirwa chePCR chinopisa.Sezvo tembiricha inokwira, iyo yakapetwa kaviri-yakasungwa amplification chigadzirwa chinonyungudika, zvichikonzera kudzikira kwefluorescence intensity.Kana imwe tembiricha (Tm) yasvika, huwandu hukuru hwezvigadzirwa huchanyungudika.Fluorescence inodonha zvakanyanya.Zvigadzirwa zvePCR zvakasiyana zvine maTm akasiyana uye akasiyana tembiricha yekunyungudika, kuitira kuti iyo chaiyo yePCR ionekwe.
Melting curve (derivative curve)
Iyo yekunyunguduka curve inotorwa kuti igadzire peak mepu, iyo inogona kunyanya kuratidza mamiriro ePCR zvigadzirwa zvimedu.Sezvo tembiricha yakanyunguduka ndiyo kukosha kweTm kwechidimbu cheDNA, mamwe maparameter anobata kukosha kweTm kwechikamu cheDNA anogona kutongwa, senge saizi yechidimbu, GC content, etc. Kazhinji kutaura, maererano neyedu primer design nheyo,kureba kwechigadzirwa chakasimudzirwa chiri muchikamu che80-300bp, saka tembiricha yekunyunguduka inofanira kuva pakati pe80°C ne90°C.
Kududzirwa kwekunyunguduka curve: Kana iyo chete yepamusoro peak inoratidzika pakati pe80 ° C-90 ° C, zvinoreva kuti fluorescent quantitative PCR yakakwana;kana iyo yepamusoro peak ichionekwa pakati pe80 ° C-90 ° C uye akasiyana-siyana epamusoro achionekwa ari pasi pe80 ° C, iyo primer dimer inotariswa zvakanyanya.Unogona kuedza kuwedzera annealing tembiricha kuigadzirisa;kana iyo huru yepamusoro ichionekwa pakati pe80 ° C-90 ° C, uye iyo yakasiyana-siyana yepamusoro inooneka zvakare kana kutonhora kuchikwira, inofungidzirwa kuti pane kusvibiswa kweDNA, uye DNA inoda kubviswa panguva yekutanga yekuedza.
Ehe, pachine mamwe mamiriro asina kujairika, ayo anozoputswa imwe neimwe pazasi.
3. Ruzivo rwepamusoro
Kuita qPCR, ndinofanira kutaura MIQE,Minimum Informationfor Publication ofQuantitativeReal-Nguva PCRMiedzo - iyo shoma ruzivo rwekuburitsa zvinyorwa nezve chaiyo - nguva yakawanda PCRmiedzo .Kuti tirerutsa kunzwisisa kwemunhu wese, ticharerutsa zvakakosha zvirimo.
Unogona kutsvaga zvinyorwa zvekutanga zveMIQE paInternet, uye chinonyanya kukosha ndechekuti inotauradata chekuongorora chinoda kupihwa pakuburitsa chinyorwa .
Vaongorori vanogona kutonga kunaka kwekuyedza nekuverenga izvi zvakadzama;vaverengi venguva yemberi vanogona zvakare kushandisa izvi kudzokorora kana kuvandudza chiedzo.
Zvakakosha kuziva kuti mune iyi runyorwa, kukosha kwechinyorwa chega chega kunoratidzwa neE kana D zvakateerana.Zvinorevei?E: ruzivo rwakakosha (runofanira kuendeswa);D: ruzivo runodiwa (kupa zvakanyanya sezvinobvira).
MIQE (1)—Kuedza Magadzirirwo
Vazhinji scumbags vakapedza kudzivirira kwavo mushure mekupedza zvidzidzo zvavo zvekupedza kudzidza havazoziva kugadzira kuyedza vakazvimirira, kuvhura zvinyorwa zvavo, uye kuita izvo mudzidzisi anovaudza kuti vaite.Nekuda kweizvozvo, dhizaini yekuyedza haina kuomarara, uye dhipatimendi rekupepeta remagazini rakati ivo vaida kugadzira mufananidzo uyu nemufananidzo, saka vakazviita vachishamisika.Aya ndiwo magadzirirwo anoitwa marara!
Pedyo nepamba, musimboti wekutanga wekuyedza kuonakuomarara kwemaitiro ekuedza.Chinhu chinonyanya kukosha ndechekuedza dhizaini, uye chinhu chinonyanya kukosha pamusoro pechiyedzo dhizaini inzira yekuseta iyo inotarirwa sampuli, referenzi sampuli (kutonga), uye nhamba yekudzokorora, kuitira kuti data rekuyedza rigone kutaurwa, kuenzaniswa, uye kugutsikana.
Muenzaniso wechinangwainoreva sampuli inoda kuti isu tione jini rinotarirwa mushure mekumwe kurapwa.Muenzaniso wereferenzindiyo sampuli isina kurapwa, iyo inowanzonzi mhando yemusango mubiology.
Experimental replicateszvakakosha zvikuru.Kazhinji, nhamba yekunyengetedza inodzokorora inofanira kunge inopfuura katatu.Izvo zvinodikanwa kusiyanisa kuti chii chinonzi biological replication uye chii chinonzi technical replication.
Biological Replicates: Muedzo wakafanana wekusimbisa wakaitwa nezvinhu zvakasiyana (nguva, zvidyarwa, mabhechi, mahwendefa ekuita).
Biological duplication
Ngatitorei mushonga wepesticide semuenzaniso.Tinoda kupfapfaidza mishonga yezvipembenene pamiti mitatu yeABC, zvino zvidyarwa zvitatu zveABC mitsara mitatu yebiological replicate, uye ndiwo muyedzo wakafanana wekuongorora unoitwa nezvinhu zvakasiyana.Asi sechiyedzo, kutonga kunodiwa zvechokwadi, saka tinokwanisa kupfapfaidza rimwe remapazi echirimwa A kuti tiumbe boka rekuyedza rechirimwa A, uye kwete kupfapfaidza mamwe mapazi echirimwa A kuti aumbe boka rekutonga.Ita zvakafanana kune B naC.
Technical Replicates (Technical Replicates): Kuedza kunodzokororwa kwakagadzirirwa kudzivirira kukanganisa kunokonzerwa nekushanda, iro rinotova gomba rakapetwa rinosanganisirwa mune zvimwe chete zvinhu.Ese marapiro uye zvidzoreso zvinofanirwa kuve nekudzokorora marongero (zvishoma zvitatu) zvejini rinotangwa uye remukati rereferenzi gene.
Kudzokorora kwehunyanzvi
Tora mhiripiri yakabatwa nemishonga yezvipembenene semuenzaniso zvakare.Kuboka rekuyedza rechirimwa A, takagadzira maburi matatu ePCR e1, 2, uye 3 kune ayo anotarirwa gene uye yemukati referensi gene zvichiteerana, kuti titore avhareji mushure mekuonekwa.Nokuda kwekutonga kwechirimwa A Mapoka anobatwawo nenzira imwecheteyo.Saizvozvo, ita kurapa kwakafanana kune B uye C zvirimwa.Uku ndiko kudzokorora kwehunyanzvi.
Zvakakosha kucherechedza izvozvochinopinda munhamba ndeyekudzokororwa kwezvinhu zvipenyu, uye kudzokororwa kwehunyanzvi ndeyekuongorora kana paine chero zviitiko zvisingaite mukuyedza maitiro, kuitira kuti mhedzisiro yekuedza ivimbike, ndiko kuti, kudzivirira zvikanganiso nekutora avhareji yavo sezvatinowanzo taura.
Negative Controls-NTC uye NRT
NTC (No-Template Control), chidzoro chisina template, chinoshandiswa kuona kana zvinhu zvekuyedza zvakasvibiswa.Kazhinji, mvura inoshandiswa se template.Kana paine fluorescent reaction, inoratidza kuti nucleic acid kusvibiswa kwakaitika murabhoritari.
Kusvibiswa uku kunobva: mvura isina kuchena, mareagents asina kukwana ane endogenous DNA, primer kusvibiswa, kusvibiswa kwemidziyo yemurabhoritari, kusvibiswa kweaerosol, nezvimwewo, zvinoda kushandisa RNase scavengers uye RNase inhibitors.Aerosol kusvibiswa ndiyo yakanyanya kuoma kuwana.Fungidzira rabhoritari yako yakaita sehutsi, ine nucleic acids dzakasiyana-siyana dzakarembera mumhepo.
NRT (No-Reverse Transcriptase), iyo inodzora isina reverse transcription, ndiyo isiri-reverse yakanyorwa RNA sekutonga kwakashata, inova kutonga kwegDNA zvakasara.
Paunenge uchiita gene expression, huwandu hweRNA hunoonekwa nekuona huwandu hwecDNA mushure mekudzoserwa kunyorwa.Kana paine gDNA yakasara kana RNA yacheneswa, zvinokonzeresa zvikanganiso mumhedzisiro yekuyedza, nekuti mhedzisiro chaiyo inowanikwa gDNA uye cDNA.Payero yeaggregate, kwete cDNA chete, gDNA inoda kubviswa zvachose panguva yekutorwa kweRNA.
MIQE (2) - ruzivo rwemuenzaniso
Iyo inonzi sampuli ruzivo inoreva kuti kana isu tikaburitsa chinyorwa nezve qPCR, isu tinofanirwa kutsanangura ruzivo rwemuenzaniso zvakajeka, chinova chikamu chakakosha chechinyorwa.Saizvozvo, kana isu tichigadzira masampuli, isu tinofanirwawo kutonga mashandiro edu kuti tive nechokwadi chechokwadi chemasampuli.
Tsanangudzo yemuenzaniso inongove mhedzisiro, uye isu tinofanirwa kubhadhara zvakanyanya kune zvakatorwa panguva yese yekuyedza.
Kusarudzwa kwezvinhu zvekuedza
Ropa sampuli - sarudza ropa idzva, kwete kupfuura maawa mana.Sero samples - sarudza kuunganidza masero matsva munguva yekukura kwakasimba.Animal Tissue-Sarudza nyowani, inokura zvine simba nyama.Plant Tissue - Sarudza nyowani, diki tishu.
Iwe unofanirwa kunge waona kuti pane izwi rakakosha mumitsara mishoma iyi: nyowani .
Kune masampuli ari pamusoro, yakanakisa, inodhura-inoshanda, uye yakagadzikana kit pamusika ndeye Foregene's kit, iyo inogona nekukurumidza uye nyore kubvisa yavo DNA neRNA.
Dyara Yese RNA Isolation Kit Plus
Kuchengetwa kwezvinhu zvekuedza
Kazhinji kutaura, isu hatikurudzire kuchengetedza samples, kana mamiriro achibvumidza.Nekudaro, kune shamwari dzakawanda dzisingakwanise kuita ongororo pakarepo mushure mesampling, uye dzimwe dzinotoda kutakura matangi enitrogen emvura kumunda kuti atore sampling.
Kune rudzi urwu rwekushanda nesimba shamwari, ndinogona kungotaura kuti haunzwisise zvinodyiwa zve reagent.Iye zvino makambani mazhinji anobatika anogadzirwa anogadzira mareagents anogona kuchengeta RNA samples pane tembiricha yekamuri, uye iwe unogona kusarudza kuishandisa.Nzira yakajairwa yekuchengetera imvura ine nitrogen kuchengetedza, uchishandisa diki mvura nitrogen tank iyo iri nyore kutakura.Mushure mokudzosa sampuli murabhoritari, ichengetedze mu -80 ° C mufiriji.
Kune zviedzo zvinosanganisira RNA, iwo matanhatu-mazwi musimboti unofanirwa kuteverwa:tembiricha yakaderera, hapana ma enzymes,uyefast .
Pfungwa yekushisa kwakadzika iri nyore kunzwisisa;pasina ma enzymes, RNase iri kwese kwese munyika yatinogara (zvikasadaro ungadai wakaurayiwa neHIV), saka kudzivirira sei RNase paunenge uchiita miedzo ipfungwa yakakosha kwazvo;kutsanya,Hakuchina Kung Fu munyika isingatyoke, kungomhanya chete hakugone kutyorwa.
Naizvozvo, neimwe nzira, iyo ipfupi nguva yekubvisa, iyo kit iri nani.Sei achidaroForegene's kit kusimbisa kukurumidza, nokuti vanozviziva zvakanaka.
PS: Vamwe vasikana vanoedza zvakanyanya, asi ivo havana kunaka senge slam dunk mushure memakore akati wandei ebasa.Vanofunga kuti Mwari haana kururama, anonyunyutira vamwe, uye ari kutsvaka upenyu.Chokwadi haana kuzvinzwisisa.Haana kudzivirira iyo RNA zvakanaka, uye slam dunk player yaive nimble.Paakanga achiita ongororo, akafunga kuti achapedza slam dunk nekatatu, kashanu uye zvikamu zviviri, asi akaita muedzo zvakanaka.
Cherechedza: Zvishoma, mikana yakawanda yekupinda kweRNase.Nzira yekuzvidzivirira sei kuti uve nekukurumidza?Hapana nzira, ingodzidzira zvakawanda.
Pakuedza kwakasiyana uye masampuli akasiyana, zvichiri kudikanwa kuverenga mamwe mabhuku uye kusarudza nzira yakakodzera yekugadzirisa.Yemuenzaniso wekuunganidza nekuchengetedza maitiro, MIQE inoda kuti inyorwe zvakajeka mubepa, kuitira kuti vanoongorora vatarise kuvimbika kwebepa, uye zvakare zviri nyore kune vechidiki vakakatyamadzwa kuti vadzokorore kuyedza kwako.
Kunyange zvazvo kuedza kwezvinhu zvipenyu kwakaoma, ndeyepamusoro-soro.Kana ukasangwarira, unogona kupidigura nyika.Semuenzaniso, kugadzira SARS mudambudziko rebiochemical, kana kugadzira mupunga wehybrid kuchengetedza vanhu bhiriyoni 1.3.Mufananidzo uri pazasi kuyedza kemikari, iwe unofanirwa kunzwisisa kuti unodada sei netsvakiridzo yako nekungotarisa kutaridzika kwake-kufanana.Kanganwa, usamusvibise.
MIQE (3) - nucleic acid kutorwa.
Nucleic acid extraction chiitiko chikuru, uye ese mamorekuru biology kuyedza anotanga ne nucleic acid kutorwa.Chekutanga pane zvese, ngatitevedzere zvirimo zveMIQE pane nucleic acid kubviswa.
Kutarisa pane iyi fomu, haugone kuramba uri pamusoro.Chimiro idzidziso.Kuti uve mudzidzi wepamusoro, unofanirwa kubvunza kuti sei.Izvo zvakakosha zvemukati tafura iyi ndezvi: Teverakuchena, kutendeseka, kuwirirana, uye kubviswa kwehuwandu hweRNA .
Chikamu chekutanga cheprocess kana chiridzwa ndicho nhanho yekubvisa nucleic acid.Kana iwe ukashandisa otomatiki nucleic acid extractor kubvisa (yepamberi, ndapota ndibatei kuti nditenge), unofanirwa kuratidza zita remuenzaniso wechiridzwa.
Zita rekiti uye
kuti ndeipi kit yakashandiswa kutsanangura shanduko, ndeapi mareagents akawedzerwa kana kuti ndeapi mavhiya akaitwa anofanirwa kutsanangurwa zvakajeka kuitira kuti vamwe vagone kudzokorora kuedza kwako.
Vamwe vanhu vanowedzera mamwe mareagents akakosha kana vachitora sampuli dzakakosha, vachifunga kuti ichi ndicho chombo chavo chakavanzika uye usaudze vamwe.Ndichiri kuchengeta zvakavanzika, ivo vanorasikirwawo nemukana wekuita kuti chinyorwa chako chipenye.Usave wakachenjera, iwe unofanirwa kuva wakatendeseka kupfuura nyika yekare Zhang mukutsvakurudza kwesayenzi, kana iwe uchida kuva wakachenjera, chinyorwa chichakuita iwe benzi.
inofanira kuyeuka nhamba yechigadzirwa chekitikana iwe ukaraira kit uye kunyora chinyorwa .Pane kazhinji nhamba mbiri pakiti: Katsi-katalogi nhamba (nhamba yechigadzirwa, nhamba yechinyorwa), Roti-yechigadzirwa nhamba (Inoshandiswa kuratidza iro batch chigadzirwa chakabva).
Uye zvakare, iyo nhamba yeCAS inowanzoshandiswa painoraira biochemical reagents, uye ini ndichaikurumbira pamwe chete.Nhamba yeCAS ndiyo nhamba yakapihwa neAmerican Chemical Society kune yega yega mushonga mutsva wemakemikari.Kazhinji, nhamba nhatu dzakabatanidzwa nedash.Rushui's CAS nhamba: 7732-18-5.Makemikari anowanzo ane akawanda aliases, asi nhamba yeCAS yakasiyana.Paunenge uchiraira mushonga, unogona kutarisa nhamba yayo yeCAS kutanga.
Pedyo nekumba, sei tichifanira kutsanangura zvinhu izvi zvakajeka?Muchokwadi, zvakare ndeyekutarisa kunaka kweRNA kubviswa.Kushandiswa kwezviridzwa uye kits kuchaita kuti RNA iwedzere kuenderana.Iyo yekubvisa chiyero cheakajairwa marabhoritari haina hombe, uye inogona kuwanikwa nemakiti.
Iwo ruzivo rweDNase kana RNase kurapwa
Nyaya yakakosha yefluorescent quantitative PCR ndeyekudzivirira kusvibiswa kweDNA, uye usaedze kana paine kusvibiswa.Naizvozvo, zvakakosha kuti utaure nzira yawakashandisa kugadzirisa DNA, kuitira kuratidza kuti DNA iri mukuyedza yakabviswa zvachose uye zvachose.inomiririrwa nedhiyagiramu yechirongwa.
Schematic diagram yeRNA neDNA
Kazhinji, nzira yekubvisa DNA ndeyekurapa RNA neDNase mushure mekubviswa.Zvisinei, idzi inzira dzekare.Commerce RNA extraction kits yakakwanisa kubvisa DNA panguva yekubvisa pasina kuwedzera DNase.Semuenzaniso, mutsara wemakiti kubva kuForegene.
Cherechedza: Kubvisa DNA panguva yeRNA kutorwa ipfumo rine ngozi rakapetwa kaviri, iro rinowedzera nguva yekushanda yeRNA kubviswa uye kuwedzera njodzi yekuparara kweRNA.Chaizvoizvo, iko kutengeserana pakati peRNA goho uye kuchena.
Pamusoro pezvo, huwandu hweDNase hwakawedzerwa kune silica-based adsorption column idiki kwazvo, uye yemhando yepamusoro DNase inofanirwa kushandiswa kuita mhedzisiro.Unoptimized DNase haigone kugaya nekukurumidza uye zvakakwana.Uyu muyedzo wechikamu chehunyanzvi hwemutengesi.Ehe, kune vakatonyanya kushamisa vatengesi vanozvirumbidza kuti DNA inogona kubviswa pasina DNase.Zvinogona kutaurwa kuti chero munhu anozvirumbidza kuti DNA inogona kubviswa zvachose pasina DNase ihooligan.DNA chimiro chine tambo mbiri dzakagadzikana, uye haigoni kudzimwa nekungotaura nekuseka.
Kuongororwa kwekusvibiswa
nzira yekuongorora: electrophoresis kuona, 1% agarose, 6V/cm, 15min, kurodha 1-3 ul
Nucleic acid quantitative analysis
inowanzoyerwa uchishandisa UV spectrophotometer.Rega nditange ndazivisa zvinorehwa nehunhu hutatu hweOD260, OD280, uye OD230.
·OD260nm: Ndiko kunyura kwewavelength kwepamusoro-soro kunyura kwenucleic acid, uye iyo yakanyanya kuyerwa kukosha inotangira pa0.1 kusvika 1.0.Kana zvisiri, dzikisa kana kuisa pfungwa pamuenzaniso kuti uuise mukati mehuwandu.
OD280nm: Ndiko kunyura kwewavelength kwepamusoro-soro kunyura kweprotein uye phenolic zvinhu.
OD230nm: Ndiko kunyura kwewavelength kwepamusoro-soro kunyura kwemacarbohydrates.
Tevere, ngatitaure nezve basa rechiratidzo chega chega.Kune A260, inogona kushandiswa kuyera goho re nucleic acid.Apo OD260=1, dsDNA=50μg/ml, ssDNA=37μg/ml, RNA=40μg/ml.
Nekuchena, tinofanirwa kutarisa mareshiyo atinowanzo kuona: OD260/280 uye OD260/230.
DNA Yakachena: OD260/280 inenge yakaenzana ne1.8.Kana yakakura kudarika 1.9, inoratidza kuti kune kusvibiswa kweRNA, uye kana iri pasi pe1.6, inoratidza kuti kune mapuroteni uye phenol kusvibiswa.
· Yakachena RNA: 1.7
·OD260/230: Ingave DNA kana RNA, kukosha kwereferensi i2.5.Kana iri pasi pe2.0, inoratidza kuti kune kusvibiswa kweshuga, munyu uye organic matter.
RNA kuvimbika
Zvakakosha kuyera kuvimbika kweRNA.Kazhinji, zvinodikanwa kuita RNA denaturation gel kuyedza kutarisa kana kupenya kuri pakati pe28S ne18S RNA hukama hwakapetwa kaviri.Kana bhendi rechitatu 5S richionekwa, zvinoreva kuti RNA yatanga kuderera, kunze kwezvipembenene.
Dhata yeRNA yemhando yekuongorora: Pamusoro peiyo bvunzo dziri pamusoro, kune zvakare mamwe epamusoro chiridzwa bvunzo maererano neRNA kutendeseka, senge RQI kutendeseka bvunzo yeExperion otomatiki electrophoresis system, iyo inogona kuona kana RNA yakaderedzwa zvisingaonekwe.
Mukutsvagisa kwesainzi, fluorescent quantitative PCR kuenzanisa pakati peiyo inotarirwa gene uye yemukati referensi gene.Naizvozvo, mukuita kweRNA sampuli kuchengetedza, RNA kudhirowa, nezvimwewo, chinangwa chikuru ndechekuona kutendeseka kweRNA.
Kutendeseka kweRNA kunokanganisa sei kuenzana pakati peiyo inotarirwa gene uye yemukati referensi gene inogona kunzwisiswa zviri nyore kubva pamufananidzo uri pazasi.Kushatisa kuchaita kuti gene kusakwana, kungave kusakwana kweiyo yemukati referensi gene kana kusakwana kweiyo inotarirwa gene, ichave nekukanganisa kukuru pane data.
Schematic dhayagiramu yechinangwa gene uye referenzi gene, haifanirwe kuve yechokwadi
Inhibition bvunzo (ingave iyo CT kukosha yakadzvanywa pasi pepamusoro kana yakaderera kusungwa kana mamwe mamiriro)
Tichitora mufananidzo uyu semuenzaniso, tsika dzeCt dzema curves mashanu ndeaya anotevera.Kugoverwa kwemaitiro eC CT pakati pemakumbo haana kuenzana, uye maitiro eCt anononoka pasi pepamusoro uye pasi, iyo ndiyo nyaya yePCR inhibition.
Chinhu chakakosha: Mukuita kubviswa kweRNA, isu tinofanirwa kusiya pfungwa dzisiridzo togadzira dzakaringana.
Pfungwa isiriyo ndeiyi: Kubvisa RNA kunongotevera goho, uchifunga kuti yakakura huwandu hweRNA inowanikwa, zviri nani.Muchokwadi, kana tichiita quantification, kana huwandu hwemajini husina kunyanya kukura, hatidi RNA yakawanda.Huwandu hweRNA hwaunobvisa hunotokwana.
Pfungwa chaiyo ndeiyi:RNA kubviswa kunofanirwa kutevedzera kuchena, kutendeseka uye kuenderana.Kuchena kunogona kuve nechokwadi kuti iyo inotevera reverse transcript haina kuvharwa uye iyo data haizokanganiswa neDNA.Kutendeseka kunovimbisa kuenzana kwezvinangwa zvakatevedzana uye mareferensi emukati.Consistency inovimbisa kugadzikana sampuli kurodha.
MIQE (4) - reverse transcription
Kusanzwisisa: kutsvaga kwehuwandu hwemhando yepamusoro.
Pfungwa yakarurama: Tevedzera kusimba (kugadzikana), zvisinei nehuwandu hweRNA yakatakurwa, kushanda kwereverse transcription kunoramba kuripo, kuve nechokwadi kuti kusiyana kwecDNA kunogona kunyatsoratidza kusiyana kwemRNA.
Isu tinotsanangura maitiro aya nedhayagiramu yechirongwa:
Schematic dhayagiramu ye reverse transcript kunyatsoita, usave chokwadi
Chekutanga pane zvese, isu tinofanirwa kunzwisisa mutsauko pakati peiyo reverse transcription process uye PCR process.PCR inopinda maitiro akawanda ekudziya uye annealing, uye chidimbu chinotarirwa chinokura zvakanyanya;nepo reverse transcription isina maitiro aya, tinogona kufungidzira kuti reverse transcription ndeye-kune-imwe Panguva yekudzokorora, sezvidimbu zvakawanda zveRNA.
sezvo kune vanogona kuwana zvidimbu zvakawanda zvecDNA Information, inofanira kunzwisiswa ikozvino, nokuti zvidimbu zvakakura uye zviduku zvakadzoserwa-reverse-yakanyorwa, uye hazvibviri kutarisa pane chimwe chidimbu.Uye nekuti huwandu hweRNA hudiki, huwandu hwecDNA hwakawanikwa hudiki, kusiyana nePCR, iyo ine kusimudzira, saka hazvigoneke kuona.
cDNA electrophoresis zvawanikwa
Chechipiri, zvine musoro, reverse transcription inoitwa one-to-one, asi hapana reverse transcriptase kubva kune chero kambani inogona kuita izvi.Chaizvoizvo, kugona kweakawanda reverse transcriptases kunodzungaira pakati pe30-50%.Kana zviri izvo, isu tingada kuve neyakagadzikana reverse transcript kunyatsoita, izvo zvatiri kuda kuona mumufananidzo: 3 RNAs inowana 2 cDNAs, 6 RNAs inowana 4 cDNAs, saka zvisinei nekuti yakawanda sei sampuli yakatakurwa, iyo reverse transcript inoshanda yakagadzikana.Hatidi kuona mamiriro ekuti reverse transcription performance haina kugadzikana uye kukwirira kwakanyanya kunodzivirirwa.
Saka, ungaona sei kuti reverse transcription performance yakagadzikana?Iyo nzira iri nyore kwazvo, iwe unongoda kuita bvunzo yekuenzanisa: imwe ndeye kudzoreredza kunyora mucDNA mushure mekupeta kaviri dilution yeRNA, uye imwe ndeyekuita kaviri dilution mushure mekudzoreredza kunyora mu cDNA, uye wozoita qPCR kuti uone kutsetseka kwakawanikwa Zvinoenderana.Semudzidzi wepamusoro, unofanirwa kuzvinzwisisa mumasekonzi.Sezvinoratidzwa pasi apa:
Dilution yeRNA uye cDNA yekuyedza kana kushanda kwe reverse transcription kwakagadzikana
Reverse transcriptase uye kit
Iyo yakakwana sei fluorescent yehuwandu PCR iine yakanakisa reverse transcriptase uye kiti.Reverse transcriptase yakakamurwa kuita mhando mbiri zvichienderana nekwabva, AMV kanaM-MLV, uye maitiro avo akafanana neanoratidzwa mutafura.
RNase H chiitiko
RNase H iRibonuclease H, zita reChinese ribonuclease H, inova endoribonuclease inogona kunyatso hydrolyze RNA muDNA-RNA hybrid cheni.RNase H haigoni hydrolyze phosphodiester zvisungo mune imwe-yakasungwa kana kaviri-yakasungwa DNA kana RNA, kureva, haigone kugaya imwe-yakasungwa kana yakapetwa kaviri DNA kana RNA.Inowanzo shandiswa mukubatanidzwa kwechipiri tambo ye cDNA.
Chinhu chinoshamisa.Isu tinoti reverse transcriptase ine RNase H chiitiko, kwete kuti reverse transcriptase ine RNase H, uye hazvigoneke kupatsanura RNase H kubva kune reverse transcriptase, pamwe nekuda kwekuumbwa kwemamwe mapoka mune reverse transcriptase Ichi chiitiko chinokonzerwa nereverse transcriptase.
Naizvozvo, zvisinei nepamusoro reverse transcription performance ye AMV, kuita kwayo RNase H kunoderedza goho re cDNA.Ehe, vanogadzira reagent vanogara vachinatsiridza zvigadzirwa zvavo kubvisa RNase H chiitiko mune reverse transcriptase sezvinobvira kuwedzera goho recDNA.
Annealing tembiricha
Sekondari chimiro cheRNA pane tembiricha dzakasiyana
Ona iyo nhamba iri pamusoro peiyo yechipiri chimiro cheRNA patembiricha dzakasiyana, uye shandisa iyo mFold online chishandiso kuona chechipiri chimiro chechidimbu chakanangwa pasi petembiricha chaiyo uye mamiriro emunyu.Pa 55 ° C, chimiro chechipiri cheRNA chichiri chakaoma zvikuru, reverse transcriptase haigoni kushanda, uye chimiro chechipiri hachigoni kugadziriswa zvachose kusvika 65 ° C, nepo tembiricha yakakwana yeAMV neM-MLV yakadzikira zvakanyanya kupfuura tembiricha iyi.
kuita sei?Chimiro chechipiri ndechekubatana kwekubatanidza kwe template pachayo, iyo inotungamirira kumakwikwi akasimba pakati pekutanga uye reverse transcriptase uye template, zvichiita kuti matambudziko akawanda akadai seE yakaderera uye kusakwanisa kudzokorora.
kuita sei?Ingowedzera tembiricha yekuvharisa zvakanyanya sezvinobvira.
Vazhinji vanogadzira reagent vari kuvandudza yavo reverse transcriptase kuburikidza nemajini engineering.Vamwe vanowedzera tembiricha yekupindura, senge Jifan naAidelai, uye vamwe vanobvisa boka rinoshanda reRNase H enzyme kuvandudza hukama pakati peiyo enzyme neRNA template.Kubatana kwepamusoro kunogona kukwikwidza kunze kwechipiri chimiro uye kuverenga zvakanaka, uye zvakare kunatsiridza zvakanyanya kushanda kwe reverse transcription.
Pfungwa inokosha: Reverse transcription inonyanya kukosha kutevera kuwirirana kwe reverse transcription performance (enzymes haifaniri kungoshanda chete asiwo yakagadzikana), panzvimbo yehuwandu hwemuenzaniso wakatakurwa, kana isiri iyo yakawanda-yakakura fluorescent quantitative PCR, hazvizoiti zvachose.Mazhinji cDNAs.
Vagadziri vakasiyana siyana vakaitawo zvimwe nhamburiko mukutsvaga kuenderana.Semuenzaniso, makambani mazhinji parizvino akarongedza reverse transcription seyakajairwa kit inotengeswa, inova sarudzo yakanaka.
Semuenzaniso, Foregene's RT Easy Series kits:
RT Easy I (Master premix yekutanga strand cDNA synthesis kit)
MIQE (5) - yakananga gene ruzivo
Mufananidzo uri pamusoro unotsanangura
1. Kuti jena iri rinoshanda here pakuedza zvinodzokororwa zvinogona kutariswa nekuedza kwakadzokororwa.
2. Gene ID, munoziva.
3. Gene urefu, hurefu hwakazara hwechinangwa chejini zvirokwazvo hapana dambudziko.Paunenge uchigadzira maprimers, ita shuwa kuti kureba kweamplicon kuri pakati pe80-200bp kuti uve nechokwadi chekusimudzira kurinani.
4. Sequence Blast kuenzanisa ruzivo, iyo inotarisirwa gene inoda kufananidzwa mu genebank kudzivirira kusiri-chaiyo amplification.
5. Kuvapo kwepseudogenes.A pseudogene iDNA sequence yakafanana nejini rakajairwa asi inorasikirwa nebasa rayo.Iyo inowanzovapo mumhuri yakawanda-gene ye eukaryotes.Kazhinji inomiririrwa ne ψ.Iyo isiri-inoshanda genomic DNA kopi mune genome iyo yakafanana neiyo coding gene kutevedzana., kazhinji hadzinyorwi, uye hadzina revo yakajeka yomuviri.
6. Position of primers inoenderana neexons uye introns.Mumakore ekutanga, patakagadzirisa dambudziko rekusvibiswa kweDNA, taigara tichitarisa nzvimbo dzemaprimers, exons, uye introns, uye taiwanzofunga kugadzira maprimers mukati memukati kudzivirira kukwidziridzwa kweDNA.Ndokumbira utarise mufananidzo uri pazasi: dema inomiririra introns, mablues akasiyana anomiririra exons, pink inomiririra zvakajairika maprimers, uye tsvuku tsvuku inomiririra intron-spanning primers.
Schematic, hazvina kumboitika zvechokwadi
Icho chirongwa chakakwana sei ichi chinoratidzika, asi muchokwadi, kazhinji, iyo trans-intron primers haisi yemashiripiti sezvainofungidzirwa, uye inokonzeresa kusiri-chaiyo amplification.Saka nzira yakanakisa yekudzivirira kusvibiswa kweDNA kubvisa DNA zvachose.
7. Kufanotaura kwemamiriro ezvinhu.Uchishandisa uyu muenzaniso zvakare, shandisa iyo mFold online chishandiso kuona chechipiri chimiro chechinangwa chechidimbu pane yakatarwa tembiricha uye munyu.
Sekondari chimiro cheRNA pane tembiricha dzakasiyana
Chimiro chechipiri ndechekubatana kwekubatanidza kwe template pachayo, izvo zvinozotungamirira kumakwikwi akasimba pakati pekutanga uye template pairing, uye mikana yekusungirirwa kweprimer ishoma, zvichikonzera kutevedzana kwezvinetso zvakadai sepasi E uye kusakwanisa kudzokorora.Kuburikidza nekufanotaura kwesoftware, kana pasina yechipiri dhizaini dambudziko, zvingave zvakanaka.Kana iripo, nyaya yedu yekutevera ichanyatso kurukura nzira yekugadzirisa dambudziko iri.
MIQE (6)—qPCR Oligonucleotides
Kune fluorescent quantitative PCR, chinhu chekutanga chaunonetseka nacho zuva rega rega RNA kudhirowa, uye chinhu chechipiri chinogona kunge chiri primer dhizaini.
Chekutanga pane zvese, isu tichiri kutarisa mitemo nezve primer dhizaini zvinoenderana neMIQE cheki.Izvo zviri nyore zvekuti scumbags anogona kuseka, uye isu tinogona kuipedzisa mumutsara mumwe: tsvaga kutevedzana uye chinzvimbo cheiyo primer probe uye nzira yekushandura.Kune iyo yekutanga nzira yekuchenesa, primer synthesis yakachipa parizvino, qPCR yakakodzera PEJI uye pamusoro penzira dzekuchenesa, uye ruzivo rwechishandiso chekugadzira hachina kukosha.Vanhu vazhinji vanga vachiita maprimers kwemakumi emakore uye havazive kuti synthesizer ndeye ABI3900.
Nezve misimboti yekutanga dhizaini, haufanirwe kuabata nemusoro, nekuti mazhinji ekutanga ekugadzira software kana maturusi epamhepo anogona kutarisira matambudziko aya (yakakurudzirwa online tool primer3.ut.ee/), uye 99.999% yeprimer dhizaini haina kuitwa nemaoko Tarisa, munyori dzimwe nguva anogadzira mazana emaprimers pazuva, kana iwe ukaverenga imwe neimwe, ichapfuura imwe neimwe.
Ingotarisa mapoinzi anotevera mushure mekunge maprimers agadzirwa:
1. Zvigadzirwa zvekugadzira zviri pedyo nekuguma kwe 3: Muchiitiko chekushandisa oligo dT primers ye cDNA yekutanga-strand synthesis, tichifunga nezve reverse transcription performance uye RNA kutendeseka, mapeji akagadzirwa anoda kugadzirwa pedyo ne3 kuguma kwekuvandudza kushanda kweamplification.Shandisa mufananidzo kutsanangura sezvinotevera (hapana nzira yekunzwisisa izvi):
Sei maprimers achifanira kugadzirwa pedyo ne3 ′ kuguma, haifanire kunge iri yechokwadi
2. TM kukosha: Nhamba yeTm iri pa 55-65 ° C (nokuti basa re exonuclease ndiro rakakwirira pa 60 ° C), uye GC zvinyorwa zviri pa40% -60%.
3. KUPUSHA: Kuti udzivise kukwidziridzwa kusiri kweiyo genome, Blast inofanirwa kushandiswa pakuwedzera ongororo.
MIQE(7)—qPCR maitiro
1. qPCR kit
Zvinoenderana nezvinodiwa neMIQE, isu tinofanirwa kutsanangura zvakajeka mamiriro ekuita akazara muchinyorwa, kusanganisira kumisikidzwa kwePCR reaction system, ndeipi kititi inoshandiswa, ndiani mugadziri, yakakura sei maitiro ekuita, ingave nzira yedhayi kana nzira yekuferefeta inoshandiswa, PCR zvirongwa zvechirongwa.Veteran vatyairi vanozoona kuti chero bedzi kiti ichisarudzwa, ruzivo rwuri pamusoro rwakatemerwa.
Parizvino, kugadzirwa uye kugadzirwa kwefluorescent quantitative PCR kits tekinoroji yakakura kwazvo.Chero bedzi iwe usingasarudze vagadziri vakaipa zvakanyanya, mukana wezvinetso hauna kukwirira, asi isu tichiri kuda kugovana newe mashoma mapoinzi:
Kupisa-kutanga Taq enzyme:Iyo inonyanya kukosha yePCR ndiyo inopisa-kutanga Taq enzyme.Iwo anopisa-ekutanga ma enzymes pamusika anowanzo kupatsanurwa kuita marudzi maviri, imwe ikemikari yakagadziridzwa inopisa-yekutanga enzyme (unogona kuifungidzira separafini inomisikidza), uye imwe yacho Ndiyo inopisa-yekutanga enzyme yekugadziridza antibody (antigen-antibody inosunga).Kugadziriswa kwemakemikari inzira yekutanga yekupisa-kutanga ma enzymes.Kana imwe tembiricha yasvikwa, enzyme inoburitsa basa rayo.Iyo antibody-yakagadziridzwa inopisa-yekutanga enzyme inoshandisa biological nzira kuvharira basa reiyo enzyme.Kana imwe tembiricha yasvikwa, masoja ekudzivirira chirwere anozoitwa denatured uye kusashanda seprotein, uye basa re enzyme rinopinzwa mukutamba.
Zvisinei, chii chiri kushandiswa kweizvi?Izvi ndizvo zvazviri, basa rekuburitsa maantibody-akagadziridzwa enzymes rinokurumidza kupfuura iro remakemikari akagadziridzwa enzymes, saka maererano nekunzwa, maantibody-akagadziridzwa ma enzymes ane mukana mudiki, zvekuti hapana chaizvo ma enzymes akagadziridzwa makemikari mumakiti pamusika.Kana iripo, saka tekinoroji yemugadziri uyu ichiri kunamatira munguva yemireniyamu.
Magnesium ion concentration:Magnesium ion concentration yakakosha muPCR reaction.Yakakodzera magnesium ion concentration inogona kukurudzira kuburitswa kweTaq enzyme basa.Kana iyo yakanyanyisa kuderera, basa re enzyme richaderedzwa zvakanyanya;kana iyo yekumisikidza yakawandisa, iyo enzyme-catalyzed isiri-chaiyo amplification inowedzerwa.Iko kusungirirwa kwema magnesium ion kuchakanganisawo kunyunguduka kwemaprimers, tembiricha yekunyungudika yetemplate uye zvigadzirwa zvePCR, zvichikanganisa goho rezvimedu zvakasimudzwa.Iko kusangana kwema magnesium ion kunowanzo kudzorwa pa25mM.Ehe, kune kititi yakanaka, kuwanda kweiyo magnesium ion kunofanirwa kudzorwa zvakanaka.Vamwe vatengesi vanowedzera magnesium ion chelating agent kune reagent, iyo inogona kuzadzisa mhedzisiro yekugadziriswa otomatiki kweiyo magnesium ion concentration.
Fluorescent dye concentration:Dhayi yefluorescent, inova SYBR Green yatinowanzo shandisa, inonyanya kugadzira fluorescence nekusunga kune diki groove yekaviri-stranded DNA, nekuti kusungirirwa kwedhayi kune kaviri-tambo DNA hakuna chaiyo, kureva kuti chero yakasungwa kaviri DNA yakasanganiswa nayo, DNA inosanganiswa neiyo template inogona kuitika, mafluores uye madhigirii anozoitika mu DNA. gadzira chiratidzo chekumashure.
PS: Nekuda kwezvimiro zvayo zvinonzwa chiedza, zvigadzirwa pamusika zvinowanzoiswa mubrown opaque centrifuge chubhu (sezvinoratidzwa pamufananidzo uri pazasi).Zvisinei, izvi zvichasangana nedambudziko.Zvakaoma kuona kuti mvura yacho inoyamwa here pakuita sampling.Panyaya iyi, Qingke ndiyo inonyanya kushandisa-mushandisi (sezvinoratidzwa pamufananidzo uri pazasi), uye chubhu inoonekera inoiswa mubhegi rerata rakajeka.Wobva waiisa mubhegi rerata, uchifunga nezvekureruka kwekunzvenga chiedza uye sampling.Iwe unofanirwa kusarudza nhamba chaiyo yechigadzirwa.TSE204 hupenyu husingadhuri, izvo zvinoita kuti ndide kudyara huswa.
Iko kusanganiswa kwedhayi refluorescent kunokosha zvakare.Kana iyo yekumisikidza yakadzikira, iyo amplification curve haizokwira mune inotevera nhanho uye haina kukwana;kana iyo concentration yakanyanyisa, inokonzeresa kukanganiswa kweruzha.Sezvo fluorescent quantitative PCR inonyanya kuenderana nehuwandu hweCT, kana kusanganiswa kwedhayi refluorescent kusina kugadziriswa zvakanaka, iyo yakaderera iri nani pane yakakwirira.Zvechokwadi, kusungirirwa kwedhayi kwakakodzera ndiko kwakanakisisa.
ROX: ROX madhayi anoshandiswa kugadzirisa kune zvakanaka-ku-tsime fluorescence chiratidzo zvikanganiso.Vamwe vagadziri vezviridzwa vanoda calibration, nepo vamwe vasingadaro.Semuyenzaniso, kushandiswa kweThermo Fisher Scientific's Real Time PCR amplification chiridzwa chinowanzoda calibration, kusanganisira 7300, 7500, 7500Fast, StepOnePlus, etc. Iyo general kit mirairo ichaitsanangura.
Foregene's qPCR Musanganiswa zvakare ine ROX dhayi, iri nyore kushandiswa mumhando dzakasiyana.
Yakaneta hydrogen bond kurapwa: Kurapwa kweasina simba hydrogen bond inyaya ine hunyanzvi.Hapana chakaverenga zvinyorwa zvemakiti akawanda, asi hapana kana mumwe wavo akataura nyaya iyi.Kutaura zvazviri, zvinokosha zvikuru.Iko kusanganiswa kwemabhesi kunonyanya kuenderana nekusimba kwehydrogen zvisungo.Akasimba ehydrogen zvisungo akajairika amplification, uye isina simba hydrogen zvisungo zvinotungamira kune isiri-chaiyo amplification.Kana isina simba hydrogen zvisungo zvisingakwanisi kubviswa zvakanaka, kwete-chaiyo amplification haigone kudzivirirwa.Mukati mechikamu chemunyori, makambani mashoma chete akaona dambudziko iri.Paunotenga kit, unogona kureva kana iwe wakafunga mhinduro mune iyi kit yaunoda kusarudza.
Reaction volume: Iyo 20-50ul system inonyanya kushandiswa, uye mavhoriyamu madiki anogona kukonzera kukanganisa.Kazhinji kutaura, iyo kit mirairo ichakurudzira kushandiswa kwePCR maitiro mavhoriyamu.Usave wakangwara uye shandisa mavhoriyamu madiki kuchengetedza mari.chinangwa che.Iro vhoriyamu inokurudzirwa nevatengesi yakanyatso kuedzwa, uye zvingave kuti havagone kugadzirisa dambudziko rekukanganisa kunokonzerwa nemavhoriyamu madiki.
2. Mugadziri uye nhamba yechinyorwa chechubhu ndiro
Wese munhu anoziva musimboti wefluorescent quantitative PCR.Fluorescence collection inonyanya kuitwa kuburikidza nePCR tube caps.Paunosarudza PCR zvinodyiwa, teerera kune maviri mapoinzi: yakanaka kutapurirana mwenje uye yakakodzera chiridzwa.Kazhinji, mabhodhi uye machubhu emhando dzakanyanya zvakanaka, asi iwe unofanirwa kusarudza nekungwarira maererano nekugadzirisa, kana zvikasadaro haugone kushandisa chiridzwa.
4. Ruzivo rwepamusoro
MIQE (8)—qPCR kusimbiswa
Ichi ndicho chinonyanya kukosha cheqPCR!Magamba mazhinji akawira mujecha umu.Ehe, zvinogoneka zvakare kuti une rombo rakanaka uye majini awakadzidza ari nyore, saka wakayangarara nemubako rechando uchitevedza mhepo.Ruzivo rwekusimbisa rweqPCR rwakaitirwa kuyedza kuvimbika kwedata.Isu tinonyora ruzivo rwakakosha rwekusimbisa sezvinotevera:
1.Specificity test
Iyo chaiyo yechinangwa gene amplification inoedzwa nekutarisa kana iyo electrophoresis mufananidzo ibhendi rimwe chete;kutevedzana kwekuongorora;kunyunguduka kuti uone kana mepu yepamusoro iri imwechete;enzyme digestion verification uye dzimwe nzira.
Pano, tinotarisa pane tiye ongororo yeasina-specific amplification achishandisa nzira yekunyungudutsa macurves.Kazhinji, kana isu tichigadzira maprimers, saizi yechimedu chechigadzirwa inodiwa kuve muhuwandu hwe80-200bp, izvo zvinoita kuti kunyunguduka kwechigadzirwa chePCR 80-85 °C.Naizvozvo, kana paine nhongonya dzakasiyana-siyana, panofanira kunge paine zvimwe zvisiri-chaiyo zvigadzirwa zvekusimudzira;kana iyo yepamusoro ikabuda pasi pe80 ° C, inowanzoonekwa seprimer dimer;kana iyo peak ikaonekwa pamusoro pe85 ° C, inowanzoonekwa sekusvibiswa kweDNA kana zvimwe Nonspecific amplification yezvimedu zvakakura.
Cherechedza: Dzimwe nguva panongova nenhongonya imwe chete pa80°C.Panguva ino, pfungwa iyi inofanira kutevedzerwa.Zvingangoita kuti mibairo yekukwidziridzwa yese ndeye primer dimers.
Yakajairika kunyungudika curve (peak imwe chete isina-yakananga amplification)
Dambudziko rekunyungudika curve (isina-chaiyo amplification yespurious peaks)
【Kuongorora nyaya】
Kune yepamusoro peak, asi iyo primer dimer yakakomba
Iyo imwechete-peak kunyunguduka curve mumufananidzo uri pazasi unogona kunyengera maziso ako zviri nyore, uchifunga kuti kuyedza kwakakwana, asi mhedzisiro yacho haina kunaka zvachose.Panguva ino, tinofanira kutarisa tembiricha yekunyungudika.Tembiricha yepamusoro iri pasi pe80°C, iri primer-dimer zvachose.
Hapana chidimbu chakatarwa, ese maprimer dimers
Apa hama yangu haigoni kumira.Mufananidzo uri pazasi ipikicha yakatorwa nenharembozha yakatumirwa kwandiri netsvina.Iwo mareagents aakashandisa ese anowanzo shandiswa mabhureki muindasitiri.Akachinja kubva kune imwe T-prefix brand kuenda kune imwe T-prefix brand.Ndofunga watozvifungira.Tsvina yakashevedzera kwandiri kuti: “Chigadzirwa chakashandiswa pamufananidzo wekutanga chakanyanya kunaka, uye chikwiriso chacho ndechemunhu.Gare gare, mushure mekushandisa reagent yawakakurudzira, inova seyechipiri mufananidzo, ine misanganiswa yepamusoro.Makandishungurudza.“
Kuparadzanisa magirafu maviri.Pakutanga kuona, imwe ine peak imwe chete, uye imwe ine peak kaviri.Nonsense, peak imwe chete yakanaka.Ichokwadi here?
Zvakaipisisa kupfuura Dou E, kana ndikaisa mifananidzo miviri mumufananidzo uri pasi apa, iwe uchanzwisisa pakarepo.Muchokwadi, isu tinoomeswa nyore nyore nemhando iyi yemufananidzo.Mushure mekunyatsoongorora, takaona kuti: iyo yepamusoro yemufananidzo wekutanga iri pa75 ° C, iyo yakazara primer dimer;iyo yepamusoro yemufananidzo wechipiri inoonekwa pa 75 ° C uye 82 ° C, zvishoma pane iyo chigadzirwa chinoratidzika.
Mifananidzo yemhinduro kubva kuvadzidzi
Saka dambudziko guru harisi dambudziko reagents, asi dambudziko rekutanga dhizaini.Panguva imwecheteyo, inoratidzawo kuti mamwe makuru makuru haasi emhando yesimbi, uye zvakare inoratidza izvo mukoma wangu akambotaura: Haisi iyo reagent brand inotsigira chinyorwa chako.Icho chinyorwa chako chakatsigira mhando yemareagents.Chimbofungidzira, kana scumbag isina kushandura reagents, data isina kururama yaizotumirwa kumagazini, uye chii chaizoitika chichava nhamo.
2. Ct kukosha kwekutonga kusina chinhu
Usatsanangure, kana iyo isina chinhu inodzora ine Ct kukosha, haisi kusvibiswa here?Nekudaro, iwe uchiri kuda kunzwisisa kuti ndeipi isina chinhu kutonga ine Ct kukosha.Kana iri NTC, zvinoreva kuti kune DNA yekune dzimwe nyika senge reagent kusvibiswa.Kana iri NRT, zvinoreva kuti RNA yakabviswa ine DNA kusvibiswa.
3. Standard curve
Kusanganisira mutsetse uye yekuverenga formula, iyo PCR inoshanda inogona kuverengerwa kuburikidza nefomati.Kuedza kwakakwana kunoda kutsetseka kweiyo curve yakajairwa kusvika pa3.32, uye R² kusvika pa0.9999.
4. Linear dynamic range
Iyo inochinja-chinja yemaitiro inoenderana.Zvinoenderana netemplate inoshandiswa kugadzira iyo yakajairwa curve, iyo dynamic range inofanirwa kusanganisira 5 concentration gradients, uye teerera kune shanduko yeCt values pahigh concentration gradients uye low concentration gradients.
5. Kuona chokwadi
Shanduko mumhedzisiro yeqPCR, ndiko kuti, kusadzokororwa zvakanaka, kureva, kusarongeka, kunokonzerwa nezvakawanda zvinhu, zvinosanganisira tembiricha, kutarisisa, uye kushanda.qPCR kunyatsoita kunowanzoita kushoma kudzoreka sezvo nhamba yekopi inodzikira.Zvakanakisa mukati-yekuedza mutsauko, iyi mutsauko wehunyanzvi unofanirwa kusiyana nekusiyana kwebiological, uye biological replicates inogona kugadzirisa zvakananga kusiyana kwenhamba mumhedzisiro yeqPCR pakati pemapoka kana marapirwo.Kunyanya kune yekuongorora assay, yakanakisa inter-assay chaiyo (kudzokororwa) munzvimbo dzese nevashandisi vanofanirwa kutaurwa.
6. Kuona kushanda zvakanaka uye LOD (mu multiplex qPCR)
LOD ndiyo yakanyanya kudzika ye95% yemasampuli akanaka akaonekwa.Mune mamwe mazwi, iyo yekumisikidzwa kweLOD iri mukati meseti yezvinangwa gene replicate haifanire kudarika 5% yekutadza kuita.Paunenge uchiita multiplex qPCR ongororo, kunyanya pakuona panguva imwe chete yekuchinja kwemapoinzi kana mapolymorphisms, multiplex qPCR inoda kupa humbowo hwekuti kurongeka kwezvimedu zvakanangwa zvakawanda hazvikanganisike muchubhu imwe chete, kuwonekwa kwakawanda uye kuona kwechubhu imwe Kushanda uye LOD inofanira kunge yakafanana.Kunyanya kana majeneti emhando yepamusoro-concentration uye akadzika-concentration target genes akawedzerwa panguva imwe chete, dambudziko iri rinofanira kubhadharwa.
Matambudziko nemhinduroKazhinji kutaura, matambudziko anowanzo sangana muqPCR debugging anotarisa pane zvinotevera zvinhu:
·nonspecific amplification
·Yakaoma sarudzo yekumisikidzwa kweprimer uye dambudziko neprimer-dimers
·Kupisa kwemvura hakuna kunaka
·Secondary chimiro chinokanganisa amplification kunyatsoshanda
nonspecific amplification
non-specific amplificationkuitika , inowanzoonekwa kana iyo primer dhizaini isina kukodzera, asi kana usiri kukurumidza kushandura maprimers, unogona kuedza nzira dzinotevera kutanga (musimboti wakabatanidzwawo):
·Kuwedzera tembiricha yemvura - edza kugadzira mabhondi ehydrogen asina simba asakwanise kuchengetedza;
·Kupfupikisa nguva yekurebesa uye yekurebesa - kuderedza mukana wekushaya simba rehydrogen bond;
·Deredzai primer concentration - kuderedza mukana wekusunga mahara ekutanga neasina kunangwa;
Low amplification kunyatsoshanda
Mamiriro akatarisana neasina-chaiyo amplification - yakaderera amplification kunyatsoita, uye matanho ekubata neakaderera amplification kunyatsoita zvinongopesana:
·Kuwedzera nguva yekurebesa nekurebesa;
· Shandura kune matatu-nhanho PCR uye kuderedza annealing tembiricha;
· Wedzera kutarisisa kwekutanga;
Ps: Vadzidzi vazhinji vakapedza kudzidza vakazvarwa muma90s havadi kudzidza maitiro ekugadzirisa zviedzo, uye vanotarisira kuti kit inogona kugadzirisa zvachose dambudziko (kana iwe uchida kuenda kukambani reagent kuti uite tsvagiridzo nekusimudzira mushure mekupedza kudzidza), chokwadi, vanogadzira reagent vanofungawo seizvi, ndinovimba ibenzi Inogona kushandiswa kana waiwana, saka vanogadzira reagent vakapedza basa rakawanda mukusagadzirisa dambudziko rekugadzirisa dambudziko rakawanda, kusanganisira kusasimba. H-bond absorption zvinhu.Kuti agadzirise dambudziko nyore nyore, mapenzi achiri kufanira kuverenga kuiswa kweiyo reagent kambani kuti aone kana paine chinhu chinotora isina simba hydrogen zvisungo.
Yakaoma kusarudzwa kweprimer concentration uye dambudziko neprimer-dimers
Nzira 1: Kazhinji kutaura, iyo kit mirairo yeqPCR yakakurudzira masisitimu uye yakakurudzirwa primer concentration.
Nzira 2: Kugadzirisa nekuisa iyo primer concentration gradient.Mufananidzo uri pazasi wakabiwa kubva kukambani kuratidza.Huwandu huri pazasi hunoratidza fluorescence huwandu hwemhedzisiro yakagadzirwa nematatu ekutanga ekusimbisa gradients (100nM, 250nM, 500nM) uye ina template yekumisikidza gradients (0.1ng, 1ng, 10ng, 100ng).Iko Ct kukosha kwemigumisiro yekuedza inorongwa sezvinotevera:
Primer Concentration Selection Concatenate imwe neimwe primer concentration mumutsara sezvinotevera:
Sarudzo yeprimer concentration iri pachena, hukama hwemutsara hwekutanga hwe100nM uye 250nM huri nani, uye hukama hwemutsara weiyo primer concentration ye500nM ishoma.Mu100nM uye 250nM, iyo Ct kukosha kwe250nM idiki, saka iyo yakakwana primer concentration ndeye 250nM.Kazhinji zvakaomesesa primer-dimers zvinogona kuoneka mukunyunguduka curve.Ko kana maprimers akagadzirwa asingakwanise kudzivirira primer-dimers?
Nzira 3: Deredza huwandu hwemaprimers uye wedzera tembiricha yekudziya (hapana chikonzero chekutsanangura).
Iko kukosha kweempirical kwekushisa kwe annealing i60 ° C.Kana iwe usina chokwadi, ungasarudza sei annealing tembiricha yakakodzera?Mhinduro yakafanana nesarudzo yeprimer concentration -gradient test.Tora mufananidzo kubva kuBio-rad kambani kuratidza dambudziko.Nekukwidziridzwa kwechimwe chimedu chinotarirwa, isa sere tembiricha gradients, imwe neimwe iine kudzokorora katatu, uye yakawanikwa yekusimudzira curve yakaita seinotevera:
annealing tembiricha sarudzo:
· 70°C, 69°C—Chaizvoizvo, ma<em>primers haakwanisi kubatanidzwa, saka hapana kukwidziridzwa.
· 67.3 ° C - Pane zvishoma zvekusimudza pakutanga, uye kukosha kweCt yakakura.
·64.5°C——Kukosha kweCt kunodzikira.
·Pa 60.7 ° C, 58.0 ° C, 56.2 ° C, uye 55.0 ° C, maitiro eCt aiwanzoita kunge akagadzikana, asi maitiro ekupedzisira efluorescence akanga akasiyana.
Nzira yekusarudza?Nheyo: Nheyo yekutanga ndiyo yakakwirira Ct kukosha.Kune imwecheteyo Ct kukosha, sarudza yakakwirira annealing tembiricha kudzivirira dimerization uye isiri-chaiyo amplification.Kunyange zvazvo kune yakakwirira yefluorescence kukosha pa55 ° C, panogona kunge kune dimers kana kuti kwete-chaiyo amplification mairi.
Asi kana iwe wakangwara sewe, iwe uchanyatso funga: Zvinonzwisisika kutaura, kana PCR reaction yakanyatsojeka, chero bedzi iyo primer concentration ichipfuura iyo diki inodiwa, iyo yakakwirira uye yakaderera mapoinzi haifanirwe kuita, senge fluorescent dhayi uye dNTPs.Chokwadi, chero tembiricha yekudziya yakagadziridzwa nemazvo, mhedzisiro yeprimer concentration paCt value inozodzikiswa.
Iyo annealing tembiricha yakagadziridzwa nemazvo, uye mhedzisiro yeprimer concentration paCT inodzikiswa
Sekondari chimiro chinokanganisa kusimudzira kugona
Ngatitorei mufananidzo kubva kuBio-rad kuratidza dambudziko.Iyo zvakare inogadzira tembiricha gradient kukwidziridza gene ine yechipiri chimiro.
Sekondari chimiro chinobuda
Zvinogona kuonekwa kuti sezvo tembiricha yekushisa inoderera, zvigadzirwa zvinotanga kuoneka uye kukosha kweCt inoenda mberi, inosvika pachiyero chiduku pa 60.7 ° C, uye apo kupisa kwekushisa kunoderera, kukosha kweCt kunowedzera.Sezvineiwo, sezvo tembiricha inowedzera, chimiro chechipiri chinovhura uye kugona kwekusimudzira kunowedzera.Mushure mekusvika kune imwe tembiricha, kuwedzera tembiricha haigone kuvandudza kugona kweamplification.Nekuti maprimers haagone kubatanidzwa zvakadzikama panguva ino.Naizvozvo,tsvaga tembiricha ine yakaderera Ct kukosha, inova tembiricha yakanakisa yekusimudzira yechipiri chimiro template!Ehe, mapenzi akangwara anofanira kuziva kuti kana zvisingakodzeri, zviri nani kushandura maprimers uye kudzivirira yechipiri chimiro dunhu.
5. Chikumbiro chepamusoro
MIQE—Data Analysis
Kuongororwa kwedata kunonyanya kupihwa nefluorescent quantitative PCR chiridzwa.Muchinyorwa chakapfuura, basa rakawanda rekuongorora data rakaitwa, rakadai sekutonga kusina chinhu, iyo yakatsanangurwa mukugadzirwa kwekuedza.Iwo emukati mareferensi majini, nhamba dzekudzokorora, nezvimwe zvakajekeswa., pano isu tinonyanya kutsanangura mashandisirwo eqPCR.
qPCR inoshandiswa zvakanyanya, uye kuongororwa kwekuyedza uye nucleic acid kuongororwa ndiyo inonyanya kushandiswa mamiriro.
absolute quantification
Logi (yekutanga kutarisisa) ine hukama hwemutsara nehuwandu hwekutenderera.Iyo yakajairwa curve inogona kudhonzwa kubva kune yakajairwa ine inozivikanwa yekutanga kopi nhamba, ndiko kuti, hukama hwemutsara weamplification reaction inogona kuwanikwa.Maererano neCt kukosha kwemuenzaniso, kusungirirwa mumuenzaniso kunogona kuverengwa.Huwandu hwematemplate ekusanganisa.
Absolute Quantitative Calculation Method
Absolute quantification inofanira kunge yakavakirwa pane yakajairwa curve.Kuti uite curve yakajairika, muyero unodiwa.Kazhinji, chiyero iplasmid inowanikwa nekugadzira iyo inotarirwa gene.Sei iri plasmid?Nekuti denderedzwa plasmid DNA ndiyo yakagadzikana zvakanyanya.Diraisa chigadzirwa chakajairwa mu5 kusvika ku6 magradients zvichienderana nereshiyo yakapetwa kaviri (10-peta dilution), uye teerera kune kufanana pakunyungudutsa.Rega kukosha kweCt kuwire pakati pe15-30.
Kugadzirira kwakajairika
Panguva imwecheteyo, sampuli inofanirwa kuongororwa inofanirawo kuderedzwa maererano (rangarira iyo dilution factor), uye kukosha kweCt kunofanirawo kuwira pakati pe15-30.Chigadzirwa chakajairwa + sampuli kuti iedzwe inoiswa pamushini pamwe chete.Mushure mekumhanya, curve yakajairwa yakagadzirwa neyakajairwa zvinhu, uye masampuli aifanira kuongororwa akaunzwa muchiyero curve kuverenga iyo yekumisikidza.
Hepatitis B virus HBV quantification ndiyo yakajairika yakakwana quantification, inogona kuverenga nhamba yekopi yehutachiona mu1ml yeropa.
Kuverenga nhamba yekopi
Sample concentration inofanirwa kuongororwa (ng/ul) = OD260 × 50ug/ml × dilution factor
Sample molecular uremu = nhamba yezvigadziko × 324
Nhamba yekopi yemuenzaniso wekuedzwa (makopi / ul) = kuwanda kweiyo sampuli kuti iongororwe / huremu hwemamorekuru emuenzaniso × 6 × 1014
Kuverengera nzira yekopi nhamba
Iri pamusoro ndiyo nzira yekuverenga yekuona huwandu.Iri idambudziko remasvomhu rinogona kugadziriswa mushure mekupedza chikoro chejunior, uye matambudziko emasvomhu anowanzo kugadziriswa nemakomputa.Kana musinganzwisise munokwanisa kuuya kuzotaurirana.
hama quantification
Relative quantification inonyanya kushandiswa mukutsvagisa kwesainzi.Ingani mavhairasi aripo mu 1ml yeropa, uye iyo hutachiona hweDNA, ichi chiitiko chakasarudzika: huwandu hweropa hunogona kutsanangurwa, uye hutachiona hweDNA hwakagadzikana.Nekudaro, zvakatiomera isu kuenzanisa nhamba yemakopi echinyorwa cheimwe gene mushizha, nekuti zvakaoma kuona saizi, uremu, uye hunyoro hweshizha, huwandu hweRNA yakabviswa hwakaoma kuona, uye kugona kwereverse transcription kwakaomawo kuziva, kureva kuti, chero nhanho inogona kuita kuti data rekuyedza rive nebugs uye harigone kushandiswa.
Naizvozvo, hukama hwehuwandu hunofanira kuunza chinhu:gene rechiratidzo chemukati .
Mune mamwe mazwi, kuenzana kwehuwandu ndeye kuenzanisa pakati peiyo inotarirwa gene uye yemukati referensi gene.Kuenzaniswa mune imwechete matishu uye sero rimwe chete, pesvedzero yehukuru hwemuenzaniso, RNA yekuwedzera huwandu, reverse transcript kunyatsoita, uye PCR kunyatsoita kudiki.Nekuda kwehukuru hukuru hwemuenzaniso, ese ari maviri emukati mareferenzi majini uye chinangwa chemajini zvakaderedzwa.Ichi ndicho chikonzero tanga tichisimbisa kufanana uye kugadzikana kare.
Internal referensi majini kazhinjimajini ekuchengetedza imba(majini ekuchengetedza imba), ayo anoreva kirasi yemajini anoratidzwa zvakatsiga mumasero ese, uye zvigadzirwa zvawo zvakakosha kuchengetedza mabasa ehupenyu ekutanga emasero.
Usavhiringa pfungwa iyi.Majini ekuchengetedza imba ndiwo mazwi ekushanda kwebiological, nepo mukati mareferensi majini ari ekuedza tekinoroji mazwi.Majini ekuchengetedza imba anofanirwa kupasa kusimbiswa asati asarudzwa semajini emukati.
Semuenzaniso, takasarudza majini akawanda ekuchengetedza imba mumufananidzo uri pazasi kuti tiedze mazinga ekutaura kwavo mumaseru akasiyana matishu, uye takaona kuti matauriro emazita e-β-2-microglobulin aive akasiyana zvakanyanya neaya emamwe matatu majini, saka aisakwanisa kushandiswa senge mukati.
Mushure mekunzwisisa basa rekugadzirisa remukati referensi gene, maviri algorithms anotorwa nekuda kwekuiswa kwemukati referensi gene.
·double standard curve method
·2 – △△Ct nzira (CT kukosha kwekuenzanisa nzira)
Kana iwe uchifarira kudzidza marudzi uye mabasa emajini, ndapota siya tsvagiridzo yealgorithms uye shandisa mafomula zvakananga, kana kushandisa michina zvakananga;kana iwe uri murume akatwasuka mumasvomhu neinjiniya, ndapota inzwa wakasununguka.
double standard curve nzira
Huwandu hwejini rinotarirwa uye jini rekuchengetedza imba yemuenzaniso wekutonga uye sampu inoedzwa kuburikidza neyakajairwa curve, wobva waverenga kukosha kwehukama zvinoenderana neformula yekuverenga, inova nhanho yekutaura.
Zvakanakira: ongororo yakapusa, iri nyore yekuyedza optimization
Zvakaipa: Pajena rega rega, denderedzwa rega rega rekuyedza rinofanirwa kugadzira curve yakajairwa
Chishandiso: Imwe yeaya maviri anonyanya kushandiswa uye anozivikanwa hukama nzira dzehuwandu mukudzidza gene expression regulation.
Iyo formula ndeyotevera:
Mienzaniso ndeiyi inotevera:
Verenga huwandu hunoenderana nehuwandu hwemhedzisiro
2 - △△Ct nzira (CT kukosha kwekuenzanisa nzira)
Zvakanakira: Hapana chikonzero chekugadzira yakajairwa curve
Zvakaipa: Zvinofungidzirwa kuti amplification kunyatsoshanda iri pedyo 100%;chiyero chekutsauka ndeye <5%, uye chiyero chakajairwa uye nekubudirira pakati peimwe neimwe amplification inofungidzirwa kuenderana;iyo optimization yemamiriro ekuedzwa yakanyanya kuoma.
Chishandiso: Imwe yeaya maviri anonyanya kushandiswa uye anozivikanwa hukama nzira dzehuwandu mukudzidza gene expression regulation.
Ehe, iyo amplification inobudirira kazhinji haigone kuve yakakwana 1. Kururamisa nzira: Kana isu tichiziva kuti iyo inotarirwa gene uye referensi gene ine yakafanana amplification kunyatsoita, asi iyo amplification inobudirira haina kuenzana ne1, ipapo 2△△Ct inogona kugadziriswa se: (1+E )－△△ kugadziridzwa . 1.95－△△Ct
Parizvino, zvirimo nezve fluorescent quantitative PCR zvasvika kumagumo.
Nguva yekutumira: Kubvumbi-06-2023
