RT-qPCR ndiyo yekutanga kuyedza yemamorekuru biology, uye munhu wese anofanira kuzviziva.Iyo inonyanya kusanganisira matanho matatu: RNA kudhirowa, reverse transcription mu cDNA, uye real-time fluorescent quantitative PCR.Hazvibatsire, chii chiri kuitika?Zvinogoneka kuti pane dambudzikochiedzo chekureverser transcription!Kunyangwe zvichiita sekunge iyo reverse transcription kuyedza inongoda kuwedzera RNA, dNTP, primers, uyereverse transcriptasekune iyo centrifuge chubhu uye sanganisa zvakanaka, asi mune chaiyo yekushanda maitiro, kuchine zvakawanda zvakawanda zvinoda kutariswa.Ngatidzidzei nezvazvo!

Nzira yekutonga sei kunaka kweRNA?
Kuti uwane cDNA, mhando yeRNA yakakosha!RNA mhando inogona kuwonekwa kunyanya kubva pamativi maviri:
(1) RNA kutendeseka:RNA kutendeseka kunogona kusimbiswa neagarose gel electrophoresis. Tichitora eukaryotes semuenzaniso, iyo yakazara yakazara RNA ine mabhandi matatu akajeka, maremu emamorekuru kubva kuhukuru kusvika kudiki ndeye 28S, 18S, uye 5S, uye 28S inopenya kaviri se18S;kana mabhendi matatu achionekwa, asi mhando yebhendi yakasvibiswa kana Diffusion zvinoreva kuti RNA yakaderedzwa zvishoma.Panguva ino, ndapota ita reverse transcription reaction nekukasira uye wedzera kuisa template nenzira kwayo;kana bhendi rine huremu hudiki hwemamorekuru kana pasina bhendi rinogona kuonekwa, iyo RNA yakaderedzwa zvachose uye inoda kudhindwazve.Agilent 2100 inoratidza kuvimbika kweRNA ine peak diagraph uye RIN kukosha.Kana iyo nucleic asidhi yakasimba, iyo yekutanga ye electropherogram yakadzikama;kana iyo nucleic acid yakasvibiswa zvakanyanya, iyo yekutanga haina kuenzana uye kuwedzera kuderera kwepamusoro kunooneka;kukosha kweRIN kunoratidza kuvimbika kweRNA, mukati mehuwandu hwe0-10, iyo yakakura kukosha, zviri nani kunaka kweRNA.Zvakanaka, iyo yakakwirira dhigirii yekuzara.
(2) Kuchena kweRNA:Chiyero cheOD260/280 chinogona kuonekwa neUV spectrophotometry.Kana chiyero cheOD260/280 chiri pakati pe1.9 ne2.1, kuchena kwakanaka kwazvo.
Residual genomic DNA inogona kutungamirira kuhuwandu husina chokwadi hwemigumisiro
Kana RNA yabviswa, iyo RNA yatinowana inogona kusanganiswa negenomic DNA (gDNA) isina kucheneswa.Naizvozvo, iyo cDNA mushure mekudzoserwa kunyorwa ichasanganiswa zvakaregDNA.Panguva yekudzikaqPCRreaction,cDNAuye gDNA inogona kukwidziridzwa panguva imwe chete, zvichiita kuti CT iwedzere kukosha, saka zvigumisiro zvingave zvakarerekera.
Saka chii chatinofanira kuita mumamiriro ezvinhu aya?Foregeneanopa mazano:
(1) Ita genome yekuchenesa pane yakadzoserwa RNA, iyo inogona kubviswa nekoramu kudhirowa panguva yekutorwa kweRNA;
(2) Bata iyo RNA yakabviswa neDNaseI , asi woigumisa neEDTA;
ye reverse transcription reagentsine genome clearing modules;

Nzira yekusarudza maprimers e reverse transcription?
Reverse transcription primers zvakare inokanganisa mhedzisiro yereverse transcription reaction.Iwe unogona kusarudza zvisina kujairika primers, Oligo dT kana gene-chaiwo maprimers ekudzoreredza zvinyorwa zvinoenderana nemamiriro chaiwo ekuedzwa:
(1) Zvinyorwa zvakananga: gene-specific primers inokurudzirwa;
(2) Zvinyorwa zvechidimbu zvirefu: Oligo dT/gene-specific primers inokurudzirwa;
(3) Zvimedu zvemukati zvezvinyorwa zvenguva refu: gene-specific primers/ random primers /random primers + Oligo dT.Kana iyo inotevera qPCR yekuedza ikaitwa, Oligo dT haigoni kushandiswa yoga, nokuti kushandisa Oligo dT chete kunogona kukonzera 3 'kuguma kusarura, zvichiita kuti zvive zvisina kururama qPCR mitsva yekuedza;
(4) miRNA: Stem-loop primers kana tailing primers inogona kushandiswa.

Inguva kangani iyo reverse transcription product cDNA inofanira kudimbutswa kuti ive quantification?
Mushure mekuwana iyo cDNA yechigadzirwa chereverse transcript, kangani iyo cDNA inofanirwa kunyungudutswa yekuyedza qPCR yakakosha.Kana iyo cDNA yekumisikidza yakakwira zvakanyanya kana yakadzikira, kugona kwekusimudzira kunogona kukanganisa.Iyo cDNA concentration inogona kuyerwa, uye inofanira kuitwa sei?
(1) The cDNA concentration of the reverse transcription product haigone kuyerwa, nokuti kuwedzera kune cDNA chigadzirwa, reverse transcription product zvakare ine reverse transcript residual Buffer, reverse transcriptase, primers, nezvimwewo, izvo zvinokanganisa mhedzisiro yekuyerwa uye kukonzera OD260/280, OD260/ 230 chiyero chechokwadi cDNA hachiratidzi.Panguva ino, dzimwe shamwari dzichati, ipapo ini ndichayera chiyero mushure mekucheneswa;pano, Forgene anoda kuyeuchidza kuti cDNA haina kukurudzirwa kucheneswa, nekuti kureba kwecDNA kunowanikwa nekudzoserwa kwakasiyana, uye cDNA pfupi icharasika mukucheneswa.
(2) Saka toita sei?Pamberi pekuyedzwa kweqPCR, iyo dilution gradient yecDNA inogona kutariswa kuburikidza nekutanga-kuyedza.Semuyenzaniso: shandisa cDNA stock solution, 10-fold dilution, uye 100-fold dilution sematemplate ekuedza kweqPCR, uye sarudza dilution factor ine kukosha kweCT muchikamu che18-28.

MiRNAs inofanira kudzokororwa sei?
miRNA imokurukuru ine tambo imwe chete RNA ine saizi ingangoita 22 nt iyo isingakodzere mapuroteni.Nekuda kwehurefu hwayo hupfupi, yakajairwa qPCR nzira yakaoma kuienzanisa zvakananga, saka kazhinji inodiwa kuwedzera miRNA;nzira dzinowanzo shandiswa reverse transcription ye miRNA dzinosanganisira stem-loop nzira uye miswe nzira.
Iyo stem-loop nzira ndeyekuwedzera iyo miRNA nekuwedzera stem-loop primers.Iyi nzira yekuona ine yakanyanya kunzwisiswa uye yakanangana, asi yekuona kubuda kwakaderera.Imwe reverse transcription inogona kungoona imwe miRNA uye referensi remukati;iyo nzira yekuwedzera muswe inoumbwa nembiri Inopedzwa nekubatana kwekuita kwema enzymes maviri, ayo ari PolyA polymerase uye reverse transcriptase.PolyA polymerase ine basa rekuwedzera PolyA miswe kune miRNA kuti iwedzere kureba kwayo, uye reverse transcriptase inoita reverse transcript reaction.Iyi nzira ine yepamusoro yekuona kuburikidza uye inogona kuona akawanda miRNAs uye mukati mareferensi mune imwe reverse transcription, asi kunzwisiswa uye hunhu hwakaderera mune stem-loop nzira.