Wese munhu ari kutaura nezve musimboti wekuyedza qRT-PCR, dhizaini yekutanga, dudziro yemhedzisiro, nezvimwe, asi ndinofunga ndinofanira kugoverana newe kuyedza kushanda kweqRT-PCR.Idiki, asi ndeye maresults.

Tisati taita qRT-PCR, tinoda kuve nekunzwisisa kwakajeka kweRNA yedu uye nzira dzekushanda.Pamusoro pazvo, nhamburiko dzedu dzine chinangwa chokuwana zvibereko, panzvimbo pokungodzidzira.Saka tisati taita qRT-PCR, tinoda kuona zvinotevera (zvimwe zvinongoshanda kuSYBR).

1 Une chokwadi here kuti RNA yako haina kuderedzwa?

NanoDrop 2000 inogona kungoona kusungirirwa uye kuchena kweRNA, asi haigone kuona kuvimbika kweRNA.

Iyo RNA (RNA Intesity Number) kukosha inogona kuratidza kuvimbika kweRNA, iyo inowanikwa neAgilent 2100 Bioanalyzer system.

Fig. Schematic dhayagiramu yeRIN tsika dzemhando dzakasiyana dzeRNA (eukaryotes)

Nekudaro, marabhoritari kazhinji haana Agilent 2100 Bioanalyzer.Muchiitiko ichi, tinogona kuona kuburikidza ne formaldehyde gel, asi chinodiwa chehuwandu hweRNA hwakakwira, saka nzira inokurumidza ndeye kushandisa yakajairwa gel electrophoresis.Inodiwa kuve munzvimbo isina nuclease-isina, saka zvinodikanwa kushambidza tangi ye electrophoresis, sol bhodhoro, gel bracket uye muzinga neDEPC mvura.Iyo agarose hainawo nuclease-isina (chero ichangobva kuvhurwa), uye Loading Buffer inofanirwa kuvhurwa ichangovhurwa zvakanyanya sezvinobvira, iine 1.2% gel.

Ziva kuti iyo gel inofanira kunyungudika zvachose, zvikasadaro ichakonzera inhomogeneous mabhandi, sezvakaratidzwa mumuenzaniso 9 mumufananidzo.Kana iyo voltage yakawandisa kana kushanda kwenguva yakareba inoburitsa kupisa uye ichikonzera kuderedzwa kweRNA, saka voltage uye nguva zvinofanirwa kudzorwa zvine musoro.Pamusoro pezvo, kumhanya kwegel kunogona zvakare kuona kana paine zvakasara zveDNA mumuenzaniso, uye tarisa kana paine huwandu hukuru hwemabhandi akachengetwa mutsime rekugovera.

Mufananidzo.Gel electrophoresis kuonekwa kweRNA

2 Une chokwadi nezve kusungwa kwecDNA yako?

Chiitiko chehama hombe murabhoritari ndechekuti iyo cDNA ye20 ul system inowanikwa nekupindurwa kwega kwega inonatswa 20X, nepo post-doctoral sisita inonatswa 10X.Ndinowanzoenderana nemamiriro ezvinhu.Nemhaka yekuti mhando yeRNA inotaurwa nemunhu mumwe nemumwe yakasiyana, mwero wekudzokera kumashure wakasiyanawo, uye tekinoroji yekudzosera inogona kunge isina kugadzikana.

Saka pese pandinowana iyo cDNA yakadzoserwa, ini ndinozotanga kuidonhedza kanenge ka3, ndozoshandisa jena rekuchengetedza imba kuita RT-PCR, huwandu hwematenderedzwa anowanzo tenderera makumi maviri neshanu, kuona iyo chaiyo yekumisikidzwa, uyezve kuona yekupedzisira dilution factor.

3 Une chokwadi here kuti maprimers ako ari nyore kushandisa?

Inogona kudarika kunyunguduka kweqRT-PCR, asi izvi zvichiri kudhura mari.Kune marabhoritari asina mari yakawanda, kana vawana maprimers akawanda, vanogona kushandisa yakajairwa RT-PCR kuona kana iri bhendi rimwe chete uye kuona kujeka kwekutanga.Kana iyo laboratori isiri kupfupika nemari, iyo chaiyo yeese maprimers inogona kuzivikanwa kamwe chete kuburikidza nekunyunguduka curve.

4 Une chokwadi here kuti yako yekuyedza mamiriro akakodzera?

SYBR inofanirwa kuchengetedzwa kubva kuchiedza chakasimba, saka edza kudzima mwenje wepamusoro paunenge uchiwedzera SYBR reagent, uye unongoda kushandisa mwenje mudiki kuti upedze.

Chengetedza SYBR pa4°C.Paunenge uchishandiswa, zvinyoronyoro pinza kumusoro uye pasi kuti usanganise zvakanaka kuti usapupuma furo, uye usabvuta zvine simba.

Dzimwe hanzvadzi diki dzinofarira kudhirowa mamakisi paPCR board nekutya kusanganisa masamples, izvo zvisiri izvo.Nekuti mamaki ako anogona kukanganisa kuunganidzwa kwemasaini efluorescent, ini ndinowanzo kurudzira vechidiki kuti vashandise zvinyorwa zvekuyedza kubatsira mundangariro, sezvakaratidzwa pazasi.

Mufananidzo.qRT-PCR sampuli yekurodha dhayagiramu

5 Une chokwadi here kuti uri kuzviita nemazvo?

Iva nechokwadi chekupfeka magirovhosi, kupfeka magirovhosi, kupfeka magirovhosi, uye kutaura zvinhu zvakakosha katatu.

Kuti uderedze kuratidzwa kweSYBR kuchiedza, ini pachangu ndinoda kuwedzera template kutanga, sezvakaratidzwa mumufananidzo uri pazasi.Zvinoenderana neruzivo, kuwedzera kwediki template kunogona kukonzera zvikanganiso zvesampling.Nokudaro, kuitira kuti kuderedze kukanganisa kunokonzerwa nekuwedzera chidimbu chiduku che template, ini ndinowanzoita kaviri sampuli zvakare, uye kaviri chiyero pakuwedzera sampuli kuderedza huwandu hweH2O2 yakawedzerwa.

Mufananidzo.Schematic dhizaini yeqRT-PCR kurodha

Wobva wagadzirisa iyo qRT-PCR sisitimu sezvinotevera.

Mufananidzo.qRT-PCR system yekugadzirira dhizaini

CHERECHEDZA: Maitiro ekugadzirisa anoda kuitwa pachando.

Mushure mekuwedzera sampuli, isa firimu rekuvhara rakajeka.Edza kusabata pamusoro peiyo yakajeka yekuisa chisimbiso firimu nemaoko ako, ingoshanda kubva panzvimbo pamativi ese efirimu.Nekuti zvigunwe zvinogona kukanganisawo kuunganidzwa kwemasaini efluorescent.Zvadaro shandisa centrifuge kukurumidza centrifuge ye 10 s pa low speed kudzivirira sampuli kubva pakurembera pamadziro.

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