Kunyatsozivikanwa kwekuonekwa
Muzviitiko zvakawanda, chinangwa cheprimer dhizaini ndechekuwedzera iyo chaiyo PCR.Izvi zvinotemerwa nekuwedzera kana kushoma kutarisika pesvedzero yezvakasiyana zvakawanda.Imwe shanduko yakakosha ndeyekutevedzana ku3′ kwekupedzisira kwekutanga.
Zvakanyanya kukosha, PCR assays akagadzirirwa hunyanzvi anowanzo kuchengetedza kushanda kwepamusoro pamusoro pehupamhi hwesimba, nekuti iyo assay haiburitse zvisiri izvo-chaiyo amplification zvigadzirwa, nekudaro kukwikwidza nePCR reagents kana kuvharira main amplification reaction.
Ehe, mune zvimwe zviitiko, chaiyo haisi iyo inonyanya kukosha, semuenzaniso, kana chinangwa chiri chekuenzanisa chakanyanya hukama asi hutachiona hwakasiyana, dhizaini yakakosha, optimization uye verification zviyero zvinodiwa.
Iyo yakanyunguduka curve inzira yakajairwa yekuongorora iyo chaiyo yemaamplicon, zvirinani maererano nekuwedzera chinangwa chimwe chete.Nekudaro, zvinofanirwa kusimbiswa kuti ma curve ekunyungudika anogona kutsausa nekuti, semuenzaniso, anogona kukanganiswa neakasanganiswa mhedzisiro ye suboptimal primers uye yakaderera template concentrations.
P5 |Iyo yakanyunguduka curve inoratidza maTm anochinja akawanikwa kubva kuviri kuwonekwa kwehuwandu hwakasiyana hwemaviri anonangwa maDNA.
A. Pazvikamu zvepamusoro (ad)), hapana chiri pachena cheprimer dimer mushure mekunge kuyerwa kweqPCR kwapera.Sezvo iyo template yekumisikidza inodzikira kusvika makumi mashanu emakopi (e), isiri-chaiyo chigadzirwa chinotanga kuoneka uye chinova icho chega chigadzirwa pane yakaderera concentration (f).
B. Muedzo wakarekodha maTms mamwechete pane zvese zvakanangwa, uye pakanga pasina pachena primer dimer kunyangwe pane yakaderera (5 makopi).Paunenge uchishandisa nzira mbiri idzi dzekuona, hapana zvigadzirwa zvekusimudzira zvakaonekwa muNTCs.
P5 inoratidza ma curves ekunyungudika anowanikwa nemasampuli umo iyo template iripo pane dzakasiyana siyana.P 5a inoratidza kuti pazvikamu zviviri zvakaderera, maTms ezvisina-specific amplification zvigadzirwa zvinogadzirwa zvakaderera pane izvo zveaamplicon chaiyo.
Zviripachena, iyi nzira yekuona haigone kushandiswa zvakavimbika kuona zvibodzwa zviripo munzvimbo dzakaderera.
Sezvineiwo, NTCs, kureva, samples dzisina DNA zvachose, hadzina kurekodha (zvisiri-chaiyo) zvigadzirwa zvekukudza, zvichiratidza kuti kumashure genomic DNA inogona kutora chikamu mune isiri-specific amplification/polymerization.
Dzimwe nguva maprimers ekumashure akadaro uye asiri-chaiyo amplification haigone kugadziriswa, asi kazhinji zvinogoneka kugadzira nzira yekuona iyo isina-chaiyo amplification mune chero template concentration uye NTC (P 5b).
Pano, kunyange kurekodha kukwidziridzwa kwechinangwa chekutarisa neCq ye35 kunoburitsa chaiyo yekumisa curve.Saizvozvo, maNTCs haana kuratidza zviratidzo zvekusanyanya kukwidziridzwa.Dzimwe nguva, maitiro ekuona anogona kutsamira padoro raamai, uye chete isiri-chaiyo amplification inoonekwa mune mamwe mabuffer enziyo, ayo anogona kunge ane hukama neMg2+ yakasiyana.
Kugadzikana kwekuonekwa
Iko optimization yeTa inhanho inobatsira mune empirical verification uye optimization maitiro ekuonekwa kweqPCR.Inopa chiratidzo chakananga chekusimba kweiyo primer yakaiswa nekuratidza tembiricha (kana tembiricha renji) inoburitsa yakaderera Cq pasina kukwidziridza NTC.
Iwo maviri kusvika mana akasiyana-siyana mukunzwa angave asina kukosha kune vanhu vane yakakwira mRNA kutaura, asi kune yekuongorora bvunzo, zvinogona kureva musiyano pakati pezvakanaka uye zvenhema mhedzisiro.
Iyo Ta zvimiro zveqPCR primers zvinogona kusiyana zvakanyanya.Mimwe bvunzo haina kusimba zvakanyanya, uye kana ikasaitwa pasi peiyo yakakwana Ta kukosha kweiyo primers, inokurumidza kudonha.
Izvi zvakakosha nekuti rudzi urwu rwekuona kazhinji runonetsa munyika chaiyo, uye kuchena kwemuenzaniso, kusungirirwa kweDNA, kana kuvapo kweimwe DNA kunogona kunge kusina kunaka.
Mukuwedzera, iyo inotarirwa kopi nhamba inogona kusiyana muhupamhi hwakasiyana, uye reagents, midziyo yepurasitiki, kana zviridzwa zvingave zvakasiyana kubva kune izvo zvinoshandiswa pakumisa bvunzo.
P6 | Iyo tembiricha gradient inoratidza kusimba kwakasiyana kwekuonekwa kwePCR.
A. Shandisa Bioline's Sensifast SYBR mastermix (catalog nhamba BIO-98050) kuita PCR pa cDNA yakagadzirwa kubva kuuropi hwemunhu RNA.
B. Shandisa chiridzwa cheBio-Rad's CFX qPCR kurekodha mepu yekusimudzira uye kunyunguduka curve ye apalene (NM_033207, F: GCCATGGAGGAAAGTGACAGACC, R: CTCATGTGTGGGTGATCTCCTAGG).
C. Girafu yekusimudzira uye kunyunguduka kwe ACSBG1 (NM_015162.4, F: CTACACTTCCGGCACCACTGG, R: GTCCACGTGATATTGTCTTGACTCAG).
D. Amplification graph uye dissolution curve yeGFAP (NM_002055.5, F: TGGAGAGGAAGATTGAGTCGCTGG, R: CGAACCTCCTCCTCGTGGATCTTC).
E. Cqs dzakarekodhwa panguva dzakasiyana tembiricha annealing, kuratidza musiyano muCq yakanyorwa pasi 7C tembiricha gradient.
P 6 inoratidza mhedzisiro yebvunzo isingadikanwi, uko qPCR yakaitwa pachishandiswa gradient Tas pakati pe59C ne67C (P 6a), vachishandisa maprimers emhando nhatu dzehuropi hwemunhu.
Zvinogona kuonekwa kubva paamplification graph kuti maOpalin primers ari kure nekunakirwa nekuti iwo maTa range akamanikana kwazvo (Mufananidzo 6b), kureva kuti, maCq akapararira zvakanyanya, zvichiita kuti maCqs afananidzwe zvakanyanya neCqs yavo yakakwana.
Iyi nzira yekuona haina kugadzikana uye inogona kutungamira kune suboptimal amplification.Naizvozvo, mbiri idzi dzekutanga dzinofanirwa kuvandudzwa.Mukuwedzera, iyo melting curve analysis (inset) inoratidza kuti iyo chaiyo yeiyi nzira yekuona inogonawo kuva yakaoma, nokuti kunyunguduka kweTa imwe neimwe yakasiyana.
Nzira yekuona ACSBG1 inoratidzwa muP 6c yakasimba kudarika nzira yekuongorora yeOpalin iri pamusoro, asi ichiri kure nekwakanaka, uye ingangodaro inogona kuvandudzwa.
Zvisinei, tinosimbisa kuti hapana hukama hunodiwa pakati pekusimba uye hutsananguro, nokuti kuputika kwemhepo kunogadzirwa nenzira iyi yekuona inoratidza kukosha kwepamusoro muTas yose (inset).
Kune rumwe rutivi, kuedza kwakasimba kunonyanya kushivirira, kubereka maCq akafanana muTas yakasiyana-siyana, sezvakaita muyedzo yeGFAP inoratidzwa muP 6d.
Musiyano muCqs unowanikwa mune imwecheteyo 8 madhigirii Celsius siyana ishoma pane 1, uye dissolution curve (inset) inosimbisa maitiro ekuona mune ino tembiricha.Izvo zvakakosha kucherechedza kuti iyo yakaverengerwa Tas uye chaiyo Ta renji inogona kunge yakasiyana zvakanyanya.
Kune mazano akawanda akagadzirirwa kubatsira vatsvakurudzi kugadzira mapeji ekutanga, mazhinji acho anobva pamitemo yakasimbiswa kwenguva refu uye kunyanya kutariswa kwakabhadharwa kune 3′ kuguma kwekutanga.Kazhinji inokurudzirwa kuisa G kana C pa3'kumagumo uye maviri G kana C mabhesi (GC clamp), asi isingapfuuri maviri ekupedzisira 5 mabhesi.
Mukuita, mitemo iyi inogona kutungamirira vatsvakurudzi, asi haisi iyo yakarurama mumamiriro ose ezvinhu.
P7 |Iyo 3′kuguma kweprimer haina mhedzisiro pane chaiyo kana kugona.
A. Nzvimbo yezvinyorwa zvemunhu HIF-1α (NM_181054.2) gene.
B. Shandisa Agilent Brilliant III SYBR Green mother liquor (Cat. No. 600882) kukudza zvinhu zvitanhatu zvekuedza.
C. Amplification girafu uye kunyunguduka curve yakanyorwa neBio-Rad's CFX qPCR chiridzwa uye 3′end primers.NTCs inoratidzwa mutsvuku.
D. Cqs rekodhi yechinhu chimwe nechimwe chebvunzo
Semuenzaniso, mhedzisiro muP 7 inopesana nemutemo we 3′end.Madhizaini ese anoburitsa zvakangofanana mhedzisiro, iine maviri ekutanga musanganiswa anotungamira kune isina-chaiyo amplification muNTC.
Nekudaro, isu hatigone kutsigira mhedzisiro yeGC clip, nekuti mune iyi kesi, kushandisa A kana T seyakakwana yemakumi matatu mabhesi hakuderedze chaiyo.
Muedzo C, panoperera F primer muGGCC, wakarekodha maCqs muNTCs, zvichiratidza kuti munhu angangoda kudzivirira kutevedzana uku pamakumi matatu.Isu tinosimbisa kuti nzira yega yekuona yakanakisa 3′ kutevedzana kweprimer pair ndeyekuongorora mamwe maprimers evamiriri.
Amplification kunyatsoshanda
Zvakakosha, kunyangwe isiri-yakananga PCR yekuona haingambove yakananga, kugona kwekusimudzira kunogona kugadziridzwa uye kukwidziridzwa nenzira dzakasiyana siyana nekushandura enzyme, doro raamai, zvekuwedzera, uye mamiriro ebhasikoro.
Kuti uongorore kushanda kwePCR yekucherechedza, zvakanakisisa kushandisa serial dilution ye10 kana 5 nguva iyo inotarirwa nucleic acid, kureva, "standard curve method".
Kana PCR amplicon kana synthetic DNA tarisiro ikashandiswa kugadzira curve yakajairwa, serial dilutions yezvinangwa izvi inofanirwa kusanganiswa nehuwandu hunoramba huri hwemashure eDNA (senge genomic DNA).
P8 |Dilution curve yekuongorora kushanda kwePCR.
A. Shandisa maprimers eHIF-1: F: AAGAACTTTTAGGCCGCTCA uye R: TGTCCTGTGGTGACTTGTCC uye Agilent's Brilliant III SYBR Green mastermix (catalog nhamba 600882) yePCR uye mamiriro ekunyunguduka curve.
B. 100 ng RNA yakadzoserwa yakanyorwa, yakaderedzwa 2 nguva, uye serially diluted cDNA samples dzakaderedzwa 5 nguva kusvika 1 ng yemunhu genomic DNA.Iyo yekunyunguduka curve inoratidzwa mune inset.
C. Iyo RT reaction, dilution, uye serial dilution yakadzokororwa yechipiri cDNA sampuro, uye mhedzisiro yakafanana.
P 8 inoratidza maviri akaenzana curves, achishandisa nzira imwechete yekuona pane maviri akasiyana cDNA samples, chigumisiro ndechekufanana, inenge 100%, uye kukosha kweR2 kwakafananawo, ndiko kuti, dhigirii yekuenzanisa pakati pe data yekuedza uye regression line kana data Degree of linearity.
Iwo maviri akajairwa macurves akafanana, asi haana kunyatsofanana.Kana chinangwa chiri chekuenzanisa chinangwa, zvinofanirwa kucherechedzwa kuti hazvigamuchirwi kupa kopi nhamba yekuverenga pasina kutsanangura kusava nechokwadi.
P9 |Kuyera kusavimbika kwakabatana nequantification uchishandisa yakajairwa curve.
A. Shandisa maprimer eGAPDH (NM_002046) kuita PCR nemamiriro ekunyunguduka.F: ACAGTTGCCATGTAGACC uye R: TAACTGGTTGAGCACAGG uye Bioline's Sensifast SYBR mastermix (katalogi nhamba BIO-98050).
B. Chati yeAmplification, curve yakanyunguduka uye standard curve yakarekodhwa neBio-Rad's CFX qPCR chiridzwa.
C. Standard curve graph uye 95% nguva yekuvimba (CI).
D. Nhamba yekopi uye 95% nguva yekuvimba yezvikamu zvitatu zveCq zvakatorwa kubva kune dilution curve.
P 9 inoratidza kuti bvunzo yakagadziridzwa, kusiyanisa kwemukati kweimwe mwero curve kunosvika kaviri (95% nguva yekuvimba, shoma kusvika pakukwirira), inogona kunge iri musiyano mudiki unogona kutarisirwa.
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Nguva yekutumira: Sep-30-2021
