FastKing RT Kit (Ine gDNase)

A-1 RNA yakashatiswa

——Chenesa yemhando yepamusoro RNA isina kusvibiswa. Izvo zvinhu zvinoburitswa RNA zvinofanirwa kunge zviri zvitsva sezvazvinogona kudzivirira kuora kweRNA. Ongorora RNA kutendeseka pane yakadonhedzwa gel pamberi peRT kuita. Mushure mekuburitswa kweRNA, inofanirwa kuchengetwa mu100% formamide. Kana RNase inhibitor ikashandiswa, kupisa kwekudziya kunofanira kuva <45 ° C, uye iyo pH inofanira kunge iri pasi pe8.0, zvikasadaro iyo inhibitor inosunungura zvese zvakasungwa RNase. Zvakare, RNase inhibitor inofanirwa kuwedzerwa mumhinduro dzine ≥ 0.8 mM DTT.

A-2 RNA ine inhibitors yekudzosera kudhindisa kusagadzikana

——Reverse transcription inhibitors inosanganisira SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, formamide, guanidine munyu, nezvimwewo Sanganisa iyo RNA yekudzora pamwe nesampuli, uye enzanisa goho nerekudzora RNA kuita kuti uone kana paine inhibitor. Shamba RNA kunaya ne70% (v / v) ethanol kubvisa inhibitors.

A-3 Yakaringana annealing yemaprimers anoshandiswa kugadzira iyo yekutanga tambo yeCDNA

—Sarudza kuti tembiricha yekuwedzera inokodzera ma primers anoshandiswa mukuyedza. Kune yakasarudzika hexamers, zvinokurudzirwa kuchengetedza tembiricha pa25 ° C kwemaminitsi gumi usati wasvika pane tembiricha yekuita. Nezve geni-yakatarwa ma primers (GSP), edza imwe GSP, kana chinja ku oligo (dT) kana zvisina kujairika hexamer.

A-4 Mari diki yekutanga RNA

- - Wedzera huwandu hweRNA. Kune RNA sampuli isingasviki makumi mashanu ng, 0.1 μg kusvika 0.5 μg acetyl BSA inogona kushandiswa mune yekutanga tambo cDNA synthesis

A-5 Chinongedzo chakanangwa hachionekwe mumatumbu akaongororwa.

—— Edza zvimwe zvinyama.

A-6 PCR maitiro anotadza

- - Kune maviri-nhanho RT-PCR, iyo cDNA template mune iyo PCR nhanho haigone kupfuudza 1/5 yeiyo reaction vhoriyamu.

A-1 Yisiri-yakatarwa annealing yemaprimers nematemplate

- - Iyo 3'-kumagumo e primers haifanire kunge iine 2-3 dG kana dC. Shandisa Gene-yakatarwa primers mune yekutanga tambo synthesis pane yakasarudzika primers kana oligo (dT). Shandisa tembiricha yakakwira yekuvharisa mumatanho ekutanga, uyezve tembiricha yepasi yekuwedzera. Shandisa inopisa-kutanga Taq DNA polymerase yePCR kuvandudza iwo chaiwo maitiro.

A-2 Yakashata dhizaini yegene-yakatarisana ma primers

——Tevera iwo iwo iwo musimboti wekusimudzira primer dhizaini.

A-3 RNA yakasvibiswa ne genomic DNA

——Rapa RNA nePCR-giredhi DNase I. Seta yekudzora maitiro pasina reverse kudhindisa kuti uone kusvibiswa kweDNA.

A-4 Kugadzira kweyekutanga dhiza

- - Dhizaina matangi asina kuenderana kwakateedzana kumagumo matatu.

A-5 Yakanyanya kukwirira Mg²⁺ kuisa pfungwa

-Wedzera Mg²⁺ kusungirirwa kwetemplate yega yega uye yekutanga kusanganisa

A-6 Yakasvibiswa neDNA yekunze

——Shandisa matipi anodzivirira maeerosol uye enzyme ye UDG.

A-1 Izvo zvemukati wechigadzirwa chekutanga chakanyanya

- - Deredza huwandu hweiyo yekutanga tambo chigadzirwa mune yakajairwa PCR yekuita nhanho.

A-2 Yakanyanya kukwirira mari muPCR reaction

- - Deredza zvinyorwa zvekutanga.

A-3 Makwikwi akawandisa

-Wedzera PCR mamiriro ezvinhu uye udzikise PCR kutenderera nhamba.

A-4 Yakadzika yakadzika tembiricha

- - Wedzera annealing tembiricha kudzivirira isiri-yakatarwa kutanga uye kuwedzerwa.

A-5 Isiri-yakasarudzika kukwidziridzwa kwezvimedu zveoligonucleotide zvinogadzirwa neDNase kudzikisira kweDNA —— Bvisa yepamusoro-soro RNA yekudzivirira hutachiona hweDNA.

RT-PCR ndeyekudzosera kunyora RNA kuita cDNA, uyezve shandisa iyo yakadzokororwa yakanyorwa cDNA se template yePCR reaction kukwidziridza chakanangwa chidimbu. Sarudza chero zvisaririra primers, Oligo dT uye geni chaiwo ma primers zvinoenderana neakasarudzika mamiriro ekuyedza. Zvese zviri pamusoro zvinyorwa zvinogona kushandiswa kwepfupi eukaryotic cell mRNA isina hairpin chimiro.

Random primer: Inokodzera RNA refu ine hairpin mamiriro, pamwe nemhando dzese dzeRNA senge rRNA, mRNA, tRNA, nezvimwe.

Oligo dT: Inokodzera RNA ine PolyA tailing (prokaryotic RNA, eukaryotic Oligo dT rRNA uye tRNA haina PolyA miswe). Nekuti Oligo dT inosungirwa kuPolyA muswe, mhando yeRNA sampuli inofanirwa kuve yakakwira, uye kunyangwe hushoma hushoma hunoderedza zvakanyanya huwandu hwakazara-kureba cDNA synthesis.

Gene-yakatarwa yekutanga: Inowirirana kune iyo template kuteedzana, inokodzera kune mamiriro apo nongedzo yekuvavarira inozivikanwa.

Pane nzira mbiri:

1. Yemukati nzira nzira: Mune dzidziso, cDNA idimbu reDNA rehurefu hwakasiyana, saka mhedzisiro yeelectrophoresis smear. Kana RNA yakawanda yakaderera, hapana chigadzirwa chinoratidza mu electrophoresis, asi izvi hazvireve kuti hapana chigadzirwa chichawedzerwa nePCR. Kazhinji, revo yemukati inogona kushandiswa kuona cDNA. Kana iro rekunongedzera remukati riine mhedzisiro, mhando yeCDNA inogona kuvimbiswa zvakanyanya (mune mashoma kesi, kana iyo yakanangwa geni chidimbu chakarebesa, panogona kuve nezvakasiyana).

2. Kana paine geni rinozivikanwa rakawedzerwa netemplate iyi, inogona kusimbiswa nezvakatanga zvei geni. Iko kukwidziridzwa kwekurehwa kwemukati hazvireve kuti hakuna dambudziko neCDNA. Nekuti zvemukati mareferenzi ane huwandu hwakawanda muCDNA, zviri nyore kuwedzera. Kana cDNA yakashatiswa zvishoma nezvikonzero zvakasiyana, kubva pakuona mukana, PCR mhedzisiro yeakaderera kuwanda chinangwa majeni anozokanganiswa zvakanyanya. Nepo mareferenzi emukati achiri akakwirira mukuwanda, kukwidziridzwa kwacho pamwe hakukanganisike.

Chikamu chakashatiswa cheRNA. Tarisa kuvimbika uye kunatsa kweRNA

Izvo zviri mukati meRNA zvemarudzi akasiyana zvinogona kunge zvakasiyana, asi kazhinji, iyo yakabviswa yakazara RNA inofanira kunge iine maviri akajeka 28S uye 18S mabhendi mune gel electrophoresis, uye kupenya kweyaimbove bhendi kunofanirwa kuve kwakapetwa zvakapetwa kaviri kupfuura kweyekupedzisira. Iyo 5S bhendi inoratidza kuti RNA yakadzikiswa, uye kupenya kwayo kwakaenzana kusvika padanho rekudzikisira. Kubudirira kukwidziridzwa kwemukati mareferensi hazvireve kuti hapana dambudziko neRNA, nekuti iro rekunongedzera remukati riri muhuwandu hwakawanda, RNA inogona kukwidziridzwa chero bedzi kukuvara kwacho kusiri kwakanyanya. OD₂₆₀/ OD₂₈₀Chiyero cheiyo yakachena RNA yakayerwa ne spectrophotometer inofanira kunge iri pakati pe1.9 ne2.1. Iko kushoma kweprotein kusachena muRNA kunoderedza huwandu. Chero bedzi kukosha kwacho kusiri kwakanyanya kuderera, RT haizokanganisike. Izvo zvinonyanya kukosha kune RT ndeye RNA kutendeka.

Kuwedzeredzwa kweiyo yemukati mareferenzi geni kunogona chete kuratidza kuti RT yakabudirira, asi hazvireve hazvo kuti zvine chekuita nemhando yeiyo cDNA strand. Nekuti zvemukati mareferenzi zvidimbu zvinowanzo kuve zvidiki muhukuru uye zvakakwirira mukutaura, zviri nyore kuti ubudirire mukudzoreredza zvinyorwa. Nekudaro, saizi uye kutaura kwechinangwa chejenisi zvinosiyana kubva kugene kuenda kujeni. Iyo cDNA mhando haigone kutongwa chete neyemukati mareferenzi kunyanya kune zvakanangwa zvidimbu zvakareba kupfuura 2 kb.

Mimwe mienzaniso ine yakaoma zvimiro zvechipiri, kana vane zvakapfuma GC zvemukati, kana zvakakosha nezvakaderera kuwanda. Mune zviitiko izvi, zvakakodzera reverse transcriptase inofanirwa kusarudzwa zvinoenderana nesaizi yechikamu chakanangwa uye nesampuli. Kune maRNA matemplate ane yakakwira GC zvemukati uye akaomarara sekondari mamiriro, zvakaoma kuvhura yechipiri chimiro pane yakaderera tembiricha, kana neyakajairika reverse transcriptase. Kune aya matemplate, Quant Reverse Transcriptase inogona kusarudzwa, sezvo kwayo reverse transcript performance iri pachena iri nani pane iyo yeM-MLV yakateedzana reverse transcriptase, iyo inogona kudzoreredza kunyora akasiyana maRNA matemplate zvakaringana uye kunyora RNA kuita cDNA yekutanga strand kusvika pakukwirira kukuru. Paunenge uchishandisa yakajairika reverse transcriptase kit, 20 μl system inongogona chete kudzoreredza kunyora 1 μg yeRNA yakazara. Ndokumbira utarise kune yakanyanya RT kugona kwe kit. Kana iyo template ikawedzerwa mukuwandisa, kudzoreredza transcript kunozofarira iyo RNA ine yakakwira yakawanda. Naizvozvo, zviri nani kuti usapfuure yakanyanya kugona yeiyo system.

A-1 Sarudza kana RNA yashoreka zvakanyanya uye kana RT ichibudirira

Pakati pese, chikonzero chekutadza kwemukati mareferenzi ekuwedzeredza chinowanzo kukonzerwa neakakomba RNA kudzikisira. Chimwe chikonzero chingangodaro kudzokorodza kutadza kuteedzera. Yemukati mareferenzi haigone kushandiswa seyakajairwa kutonga mhando yeCDNA single strand, asi inogona kushandiswa seyakajairwa kutonga kana kudzoreredza chinyorwa kuchibudirira kana pasina dambudziko reiyo RNA mhando. Chinhu chakanyanya kukosha mukutendeuka kwechinyorwa ndechekuchengetedza tembiricha yenguva dzose uye neyakagadziriswa maitiro kuitira kusimudzira maitiro ekuita.

A-2 Sarudza kana ma primers ekuwedzera emukati mareferenzi akavimbika uye kana paine chero matambudziko nema reagents anoshandiswa muPCR.

Nezvehuwandu hwehuwandu, RNA inofanirwa kuverengerwa isati yadzokororwa kudhindwa, iyo inodawo mune dzakawanda zvinodzosera kits kits, semuenzaniso, kuyera iyo RNA yekuisa se1 μg. Sezvo iyo yakadzoserwa yakadhindwa cDNA iri mhinduro yakavhenganiswa, inosanganisira RNA, oligo dT, enzyme, dNTP, uye kunyangwe diki resaiti reDNA, kutsauka kuchakonzerwa, saka hazvigoneke kunyatso kuenzanisa iyo cDNA. Naizvozvo, RNA huwandu hunodiwa. Tichifunga nezve reverse transcript kunyatsoshanda kwakaenzana pakati pemasampuli akasiyana, huwandu hweCDNA hunowanikwa hunofanirwa kunge hwakaenzana, uye iyo ongororo yekuwongorora inogona kuratidza kuenzanisa kwematanho ekutaura emageneti akasiyana muhuwandu hwakaenzana hweRNA yakazara. Paunenge uchiita yakabatana fluorescence yakawandisa PCR, yakawandisa cDNA inogona kunge isingadiwe mushure mekudzokorodza kudhindisa nekuti yemukati rejenenzi jeni rinogona kuitwa sereferenzi.

Iyo inonyanya kuenderana nemagene, uye kudzoreredza kunyorwa kwechimedu chakareba hakukwanisike kune mazhinji magene. Kutanga, kugona kwekushandurwa kwechinyorwa kwakaderera zvakanyanya kupfuura kwePCR. Kechipiri, iyo GC yakapfuma dunhu uye yechipiri chimiro chemajini mazhinji inorambidza zvese zvinodzosera zvinyorwa uye PCR. Chekupedzisira, kuvimbika nekusimudzira kugona kwePCR kunonetsa kuvimbisa panguva imwe chete. Mukuita kwekudzokorodza kudhindisa, hapana munhu anogona kuvimbisa kuwana chidimbu chakareba chemhando yepasi magene, kunyanya kushandisa oligo dT. Kana iri 5 'UTR ine GC yakawanda, zvakatooma. Naizvozvo, ichiri nzira ine musoro yekudzosera zvakanyorwa nema primers asina kujairika, tsvaga maseru maseru munzvimbo yechinongedzo, kuwedzera nezvikamu, uyezve woita iyo yekudzivirira kugaya uye ligation. Pakati pese, zvakaoma kuwedzera zvakananga zvidimbu zvakakura kupfuura 2 kb, asi hazviwanzo kugona kuwana: 1. Chekutanga pane zvese, kuvimbisa kutendeka kweRNA / mRNA, uye kubviswa kweTRIZOL kunodiwa. 2.M-MLV RT-PCR kit inogona kushandiswa zvakananga. Wedzera nguva yekuwedzera uye wedzera kutenderera nhamba mune yekuwedzeredza maitiro nemazvo. Neimwe nzira, nested PCR inogona kuiswa, kana kuita kamwe kana maviri maitiro kutanga nekukodzera kuwedzerwa denaturation uye nguva yekuwedzera pamberi peyakajairwa PCR kukwidziridzwa, izvo zvinogona kubatsira kuwedzera zvidimbu. Teerera kutendeka kwepolymerase. 3.Long Taq inogona kushandiswa muPCR kuwana mhedzisiro yakanaka. 4.Protein expression expression application, yakakura kutendeka polymerase inofanira kuiswa.

Kune maviri marudzi e reverse transcriptase anopiwa neTIANGEN: Quant / King RTase uye TIANScript M-MLV. Musiyano mukuru pakati pavo chiyero chekuisa matemplate. Quant ndeye yakasarudzika reverse transcriptase, iyo yakasiyana neinowanzo shandiswa M-MLV yakabva kuMoloney murine leukemia virus. Quant ndiyo nyowani yepamusoro-inoshanda reverse transcriptase inodzokororwa inoratidzwa neinjiniya Escherichia coli. Quant inokodzera kukwidzirisa makumi mashanu ng-2 μg yeRNA ine yakakwira reverse transcriptional chiitiko uye goho repamusoro. Inofananidzwa neyakajairika MMLV kana AMV, Chinhu chakakura kwazvo cheQuant ndechekuti ine hukama hwakasimba kwazvo neRNA matemplate uye inogona kudzoreredza zvinyorwa zvakakomba matemplate pasina yakanyanya tembiricha denaturation. Kune matemplate ane yakakwira GC zvemukati, iyo reverse kunyatsoshanda yakakwira. Nekudaro, iyi reverse transcriptase ine RNase H chiitiko, icho chingakanganisa kureba kweiyo cDNA chigadzirwa (yakakodzera ma <4.5 kb matemplate). Nezve zvakajairika kudzokororwa, TIANScript MMLV reverse transcriptase inokurudzirwa. Iyi RTase enzyme yakashandurwa ine chisina kusimba RNase H chiitiko, icho chakakodzera kwenguva refu (> 5 kb) cDNA synthesis.

Imwe-nhanho inodzosera kudhindisa uye PCR kukwidziridzwa inopedzwa mune imwechete chubhu isina kuvhura iyo chubhu inovhara pakati peCDNA synthesis nekusimudzira, izvo zvinobatsira kudzikisira kusvibiswa. Sezvo ese macDNA masampuli akawanikwa anoshandiswa mukusimudzira, iko kunzwisisika kuri pamusoro, paine hushoma hwe0.01 pg yeRNA yakazara. Kune yakabudirira nhanho-nhanho RTPCR, geni-yakatarwa ma primers anowanzo shandiswa kutanga cDNA synthesis. Iyo mbiri-nhanho nzira, kureva reverse kudhindisa uye PCR kukwidziridzwa kunoitwa mumatanho maviri. Chekutanga kudzoreredza chinyorwa kunoitwa kubva kuRNA template kuti uwane cDNA, uye iyo yakawanikwa cDNA inoiswa kune imwe kana kupfuura akasiyana PCR maitiro. Iyo nzira-nhanho nzira inogona kushandisa oligo (dT) kana yakasarudzika primers kutungamira iko kwekutanga tambo yeCDNA, uye inogona kudzoreredza kunyora zvese mRNA ruzivo kubva kune yakatarwa sampuro.