RT-qPCR inogadzirwa kubva kune yakajairwa PCR tekinoroji.Iyo inowedzera fluorescent makemikari (fluorescent dhayi kana fluorescent probes) kune yechinyakare PCR reaction system, uye inoona iyo PCR annealing uye yekuwedzera maitiro munguva chaiyo zvichienderana neavo akasiyana mwenje maitirwo.Fluorescent chiratidzo shanduko mukati mepakati inoshandiswa kuverenga huwandu hwechigadzirwa shanduko mune yega yega kutenderera kwePCR.Parizvino, nzira dzakajairika ndeye fluorescent dhayi nzira uye probe nzira.

Fluorescent dhayi nzira:
Mamwe madhayi efluorescent, akadai seSYBR Green Ⅰ, PicoGreen, BEBO, nezvimwewo, haabudise mwenje ari ega, asi anoburitsa fluorescence mushure mekusunga kune diki groove yedsDNA.Naizvozvo, pakutanga kwePCR reaction, muchina haugone kuona chiratidzo chefluorescent.Kana maitiro acho achienderera kune annealing-extension (matanho maviri-nzira) kana nhanho yekuwedzera (nhanho-nhatu nzira), tambo mbiri dzinovhurwa panguva ino, uye itsva DNA polymerase Munguva ye strand synthesis, fluorescent mamorekuru anosanganiswa mu dsDNA minor groove uye emit fluorescence.Sezvo huwandu hwePCR kutenderera hunowedzera, dhayi dzakawanda uye dzakawanda dzinosangana ne dsDNA, uye chiratidzo chefluorescent chinoramba chichiwedzerwa.Tora SYBR Green Ⅰ semuenzaniso.
Probe nzira:
Taqman probe ndiyo inonyanya kushandiswa hydrolysis probe.Kune boka refluorescent pa5 ′ kupera kweprobe, kazhinji FAM.Iyo probe pachayo ndeyekutevedzana kunopindirana kune yakananga gene.Kune boka rekudzima fluorescent pa3 ′ kupera kwefluorophore.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) Kana iyo excitation spectrum inopindirana uye chinhambwe chiri padhuze (7-10) inotora mamorekuru, donor inogamuchira iyo molecules 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.

Fig 1 Musimboti weqRT-PCR

Primer design
Mitemo

Iwo maprimers anofanirwa kugadzirwa munharaunda yakachengetedzwa yeiyo nucleic acid akateedzana uye ane chaiyo.

Zvakanakisa kushandisa cDNA kutevedzana, uye mRNA kutevedzana kunogamuchirwa zvakare.Kana zvisiri, tsvaga maCD dunhu dhizaini yeDNA kutevedzana.
Kureba kwechigadzirwa chefluorescent quantitative i80-150bp, kureba i300bp, kureba kweprimer kunowanzo kuve pakati pe17-25 mabhesi, uye musiyano uripo pakati pemaprimers ekumusoro nekudzika haafanire kunge akakurisa.

Zvinyorwa zveG + C zviri pakati pe40% ne60%, uye 45-55% ndiyo yakanakisisa.
Iko kukosha kweTM kuri pakati pe58-62 madhigirii.
Edza kudzivisa primer dimers uye self-dimers, (usaonekwe kupfuura 4 pairs of anoteedzana complementary bases) hairpin structure, kana zvisingadzivisiki, ita ΔG <4.5kJ / mol * Kana iwe usingakwanisi kuve nechokwadi chokuti gDNA yakabviswa panguva yereverse transcription Yakachena, zvakanakisisa kugadzira zvinyorwa zve intron, CG / GC , inogona kudzivisa kupfuma, AC / GC , kuguma kweA. inoenderera chimiro (2-3) yekutanga uye isiri-
yakananga The homology of the heterogeneous amplified sequence ingangoita isingasviki 70% kana ine 8 complementary base homology.
Database:
CottonFGD kutsvaga nemazwi akakosha
Primer design:
IDT-qPCR primer dhizaini

Fig2 IDT yepamhepo primer dhizaini peji peji

Fig3 mhedzisiro peji kuratidza
Dhizaini yelncRNA primers:
lncRNA:matanho akafanana nemRNA.
miRNA:Nheyo yeiyo stem-loop nzira: Sezvo ese miRNAs ari mapfupi akateedzana angangoita 23 nt, yakananga PCR kuona haigone kuitwa, saka iyo stem-loop sequence chishandiso inoshandiswa.The stem-loop sequence iDNA ine tambo imwe chete inosvika makumi mashanu nt, iyo inogona kugadzira hairpin chimiro pachayo.3 'Magumo anogona kugadzirwa seanotevedzana anoenderana nemiRNA chikamu chechidimbu, ipapo miRNA yakanangwa inogona kubatanidzwa kune stem-loop sequence panguva yekudzoreredza kudhindwa, uye hurefu hwakazara hunogona kusvika 70bp, inofambirana nehurefu hwechigadzirwa chakakwidziridzwa chakatemwa neqPCR.Tailing miRNA primer dhizaini.
Amplification-chaiyo kuwanikwa:
Online blast database: CottonFGD inoputika nekutevedzana kufanana
Kuputika kwenzvimbo: Tarisa kushandisa Blast + kuita kuputika kwenzvimbo, linux uye macos anogona kumisa zvakananga dhatabhesi renzvimbo, win10 system inogona zvakare kuitwa mushure mekuisa ubuntu bash.Gadzira yemuno blast database uye yemuno kuputika;vhura ubuntu bash pa win10.
Cherekedza: Donje rekumusoro negungwa regungwa zvirimwa zvetetraploid, saka mhedzisiro yekuputika inowanzova machisi maviri kana anopfuura.Kare, kushandisa NAU cds sedhatabhesi kuita kuputika kungangowana maviri homologous majini ane mashoma mashoma SNP misiyano.Kazhinji, iwo maviri homologous majini haagone kuparadzaniswa neprimer dhizaini, saka iwo anobatwa akafanana.Kana pane indel yakajeka, iyo primer inowanzogadzirirwa pane indel, asi izvi zvinogona kutungamirira kune chechipiri chimiro chekutanga Simba rakasununguka rinova rakakwirira, zvichiita kuti kuderera kwekuwedzera kwekusimudzira, asi izvi hazvidzivisiki.

Kuonekwa kwekutanga kwechipiri chimiro:
Matanho:vhura oligo 7 → kuisa template sequence → kuvhara pasi-hwindo → chengetedza → tsvaga primer patemplate, dzvanya ctrl+D kuti uise primer kureba → ongorora zvakasiyana-siyana zvechipiri zvimiro, zvakadai se self-dimerization body, heterodimer, hairpin, mismatch, etc. Mifananidzo miviri yekupedzisira muMufananidzo 4 ndiyo mhinduro dzebvunzo dzemaprimers.Mhedzisiro yepamberi yekutanga yakanaka, hapana chakajeka dimer uye hairpin chimiro, hapana inoenderera mberi mabhesi ekuwedzera, uye kukosha kwakakwana kwesimba remahara iri pasi pe4.5, nepo kumashure kwekutanga kunoratidza kuenderera Mabhesi matanhatu anowirirana, uye simba remahara 8.8;kuwedzera, dimer yakanyanyisa inoonekwa pa 3 'kumagumo, uye dimer ye 4 yakatevedzana mabhesi inooneka.Kunyangwe iyo simba remahara risina kukwirira, iyo 3 ′ dimer Chl inogona kukanganisa zvakanyanya amplification kujekesa uye kuita kwekusimudzira.Mukuwedzera, zvakakosha kutarisa hairpins, heterodimers, uye mismatches.

Fig3 oligo7 yekuona mhinduro
Amplification kunyatsoonekwa:
Iko kukwidziridzwa kwekuita kwePCR kunokanganisa zvakanyanya mhedzisiro yePCR.Zvakare muqRT-PCR, iko kukwidziridzwa kwakakosha kwakakosha kune huwandu hwemhedzisiro.Bvisa zvimwe zvinhu, michina uye maprotocol mune reaction buffer.Hunhu hwemaprimers zvakare hune pesvedzero huru pakuwedzera kwekuita kweqRT-PCR.Kuti ive nechokwadi chechokwadi chemhedzisiro, ese ari maviri fluorescence quantification uye absolute fluorescence quantification inoda kuona kukwidziridzwa kwemaprimers.Izvo zvinozivikanwa kuti Iyo inoshanda qRT-PCR amplification performance iri pakati pe85% ne115%.Pane nzira mbiri:
1. Standard curve nzira:
a.Sanganisa cDNA
b.Gradient dilution
c.qPCR
d.Linear regression equation kuverengera kugona kweamplification
2. LinRegPCR
LinRegPCR chirongwa chekuongorora chenguva chaiyo RT-PCR Data, inonziwo quantitative PCR (qPCR) data yakavakirwa paSYBR Green kana yakafanana chemistry.Iyo purogiramu inoshandisa isiri-yekutanga yakagadziridzwa data, inoita yekutanga kururamisa pane imwe neimwe sampu Yakaparadzana, inosarudza hwindo-ye-mutsara uyezve inoshandisa mutsara regression ongororo kuti ikwane mutsara wakatwasuka kuburikidza nePCR data set.Kubva pamateru emutsara uyu kushanda kwePCR kwemuenzaniso wega wega kunoverengerwa.Izvo zvinoreva PCR kunyatsoshanda paamplicon uye kukosha kweCt pamuenzaniso inoshandiswa kuverenga kutanga kusungirirwa pamuenzaniso, inoratidzwa mune zvisingaverengeki fluorescence units.Kupinza data uye kuburitsa zviri kuburikidza neExcel spreadsheet.Muenzaniso chete
kusanganisa kunodiwa, hapana gradient
matanho anodiwa:(Tora Bole CFX96 semuenzaniso, kwete chaizvo Machine ine yakajeka ABI)
kuyedza:chiyedzo che qPCR.
qPCR data kubuda:LinRegPCR inogona kuziva mafomu maviri ekubuda mafaera: RDML kana quantification Amplification mhedzisiro.Muchokwadi, ndiyo chaiyo-nguva yekuona kukosha kwenhamba yekutenderera uye fluorescence chiratidzo nemushini, uye iyo amplification inowanikwa nekuongorora iyo fluorescence shanduko kukosha kweiyo mutsara chikamu chekubudirira.
Kusarudzwa kwedata: Muchirevo, iyo RDML kukosha inofanira kushandiswa.Zvinofungidzirwa kuti dambudziko rekombuta yangu nderekuti software haigone kuziva RDML, saka ini ndine excel inobuda kukosha seyekutanga data.Zvinokurudzirwa kuita rough screening yedata kutanga, sekutadza kwekuwedzera samples, etc. Mapoinzi anogona kudzimwa mune yakabuda data (zvechokwadi, haugone kudzidzima, LinRegPCR inofuratira aya mapoinzi mune inotevera nhanho)

Fig5 qPCR data kunze

Fig6 kusarudzwa kwemasampuli evamiriri

Kuiswa kwedata:Vhura mibairo yemakwitsidzo.xls, → vhura LinRegPCR → faira → verenga kubva kuexcel → sarudza maparamita sezviri kuratidzwa paMufananidzo 7 → OK → baya sarudza nheyo

Fig7 nhanho dzelinRegPCR data yekuisa

Mhedzisiro:Kana pasina kudzokorora, hapana boka rinodiwa.Kana paine kudzokororwa, kupatsanurwa kunogona kugadziridzwa muboka remuenzaniso, uye zita rejini rinoiswa muchiziviso, uyezve jena rimwe chete rinozoiswa mumapoka.Pakupedzisira, tinya pafaira, tumira kunze kunze, uye tarisa zvawanikwa.Kubudirira kwekusimudzira uye R2 mhedzisiro yetsime rega rega icharatidzwa.Kechipiri, kana iwe ukakamura kuita mapoka, iyo yakagadziridzwa avhareji yekusimudza kunyatsoshanda icharatidzwa.Ita shuwa kuti kukwidziridza kushanda kwekutanga kwega kwega kuri pakati pe85% ne115%.Kana yakakurisa kana idiki, zvinoreva kuti kusimudzira kweiyo primer kwakashata.

Fig 8 Mhedzisiro uye data yakabuda

Maitiro ekuedza:
RNA mhando zvinodiwa:
Kuchena:1.72.0 inoratidza kuti panogona kunge paine yasara isothiocyanate.Yakachena nucleic acid A260 / A230 inofanira kunge yakapoteredza 2 .Kana pane kunyura kwakasimba pa 230 nm, inoratidza kuti kune zvisikwa zvehupenyu zvakadai se phenate ions.Mukuwedzera, inogona kuonekwa ne 1.5% agarose gel electrophoresis.Nongedza mucherechedzo, nekuti ssRNA haina denaturation uye molecular uremu logarithm haina hukama hwemutsara, uye huremu hwemamorekuru haugone kuratidzwa nemazvo.Kuisa pfungwa pachinhu chimwe: Nemataurirokweteisingasviki 100ng / ul, kana iyo yekuisa yakanyanya kuderera, kuchena kunowanzo kuderera kwete kureba

Fig9 RNA gel

Uye zvakare, kana sampuli yakakosha uye iyo RNA yekumisikidza yakakwira, zvinokurudzirwa kuiquote mushure mekubvisa, uye kudzikisa iyo RNA kune yekupedzisira kusungirirwa kwe100-300ng/ul yekudzoreredza transcription.Inmaitiro ekureverse transcription, kana mRNA ichinyorwa, oligo (dt) maprimers anogona kunyatsosunga kumiswe yepolyA anoshandiswa kureverse transcription, ukuwo lncRNA necircRNA zvinoshandisa random hexamer (Random 6 mer) maprimers for reverse transcription of total RNA For miRNA, miRNA-specific transcription are used for reverse transcription.Makambani mazhinji parizvino akatangisa machira akakosha.Kune iyo stem-loop nzira, nzira yekuswededza iri nyore, yakakwirira-kuburikidza, uye reagent-kuchengetedza, asi Mhedzisiro yekusiyanisa miRNAs yemhuri imwechete haifanirwe kunge yakanaka seye stem-loop nzira.Yega yega reverse transcription kit ine zvinodiwa pakusangana kwemagene-specific primers (stem-loops).Referensi yemukati inoshandiswa miRNA iU6.Mukuita kwe stem-loop inversion, chubhu yeU6 inofanirwa kudzoserwa zvakasiyana, uye kumberi uye kumashure kweU6 kunofanirwa kuwedzerwa zvakananga.Ose ari maviri circRNA uye lncRNA anogona kushandisa HKGs sereferensi yemukati.IncDNA kuwanikwa,
kana pasina dambudziko neRNA, cDNA inofanirawo kuva yakanaka.Zvisinei, kana kukwana kwekuedza kuchiteverwa, zviri nani kushandisa gene referensi yemukati (Reference gene, RG) inogona kusiyanisa gDNA kubva kumaCD.Kazhinji, RG ijeni rekuchengetedza imba., HKG) sezvinoratidzwa mumufananidzo 10;Panguva iyoyo, ndanga ndichigadzira soyabean yekuchengetedza protein, uye ndaishandisa actin7 ine introns sereferensi yemukati.Ukuru hwechidimbu chakasimudzirwa cheiyi primer mugDNA chaive 452bp, uye kana cDNA yakashandiswa setemplate, yaive 142bp.Zvadaro mhedzisiro yebvunzo yakawana kuti Chikamu che cDNA chaive chakasvibiswa negDNA, uye yakaratidzawo kuti pakanga pasina dambudziko nemhedzisiro yekurevera transcription, uye inogona kushandiswa setemplate yePCR.Hazvibatsiri kumhanyisa agarose gel electrophoresis zvakananga necDNA, uye ibhendi rinopararira, risingabvumire.

Fig 10 cDNA kuonekwa

Kutemerwa kweqPCR mamirirokazhinji haisi dambudziko maererano neprotocol yekiti, kunyanya munhanho ye tm kukosha.Kana mamwe mapeji asina kugadzirwa zvakanaka panguva yekugadzirwa kwekutanga, zvichiita kuti pave nekusiyana kukuru pakati pekukosha kwetm uye theoretical 60 ° C, zvinokurudzirwa kuti cDNA Mushure mokunge sampuli dzakasanganiswa, shandisa gradient PCR nemaprimers, uye edza kudzivisa kuisa kutonhora pasina mabhandi se TM inokosha.

Data analysis

Iyo yakajairwa hama fluorescence quantitative PCR yekugadzirisa nzira iri maererano ne2-ΔΔCT.Data processing template.

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