Ⅰ. Wedzera kunzwisiswa kweiyo reaction system:

1. Kusiyanisa RNA yemhando yepamusoro:

Kubudirira cDNA synthesis kunobva kumusoro-mhando RNA.Yepamusoro-mhando RNA inofanirwa kuve nechokwadi chenguva yakareba uye haina inhibitors isina maenzayimu ekurekodha, akadai seEDTA kana SDS.Hunhu hweRNA hunotaridza kukosha kwakanyanya kweruzivo rwekutevedzana kwaunogona kunyora kucDNA.Iyo yakawanda RNA yekuchenesa nzira idanho nzira yekushandisa isoocyanate/acidophenol.Kuti udzivise kusvibiswa kweRNase, iyo RNA yakaparadzaniswa nemuenzaniso wakapfuma muRNase (yakadai sepancreas) inoda kuchengetwa kwe formaldehyde kuchengetedza yakakwirira-mhando RNA, iyo inonyanya kukosha yekuchengetedza kwenguva refu.Iyo RNA yakatorwa kubva pachiropa chegonzo yakaderedzwa mushure mevhiki imwe yekuchengetedza mumvura, nepo RNA yakatorwa kubva mugonzo spleen yakaramba yakagadzikana mushure memakore matatu ekuchengetedza mumvura.Pamusoro pezvo, zvinyorwa zvakakura kupfuura 4kb zvakanyanya kutarisisa kuderedzwa kweRNase pane zvinyorwa zvidiki.Kuti uwedzere kugadzikana kweiyo yekuchengetedza RNA sampuli, iyo RNA inogona kunyungudika mune methalmamine yeion, uye inochengetwa -70 ° C.Thylide inoshandiswa kuchengetedza RNA haifanire kunge iine zvinhu zvakasiyana zvinodzikisira RNA.RNA, iyo inotorwa kubva pancreas, inogona kuchengetwa mumethalmamine kweinenge gore rimwe.Kana wagadzirira kushandisa RNA, unogona kushandisa nzira dzinotevera kuti uwedzere RNA: wedzera NaCl kusvika 0.2m uye 4 nguva yevhoriyamu ye ethanol, isa tembiricha yekamuri kwemaminitsi 3-5, uye 10,000 × g centrifugal kwemaminitsi mashanu.

2. Shandisa reverse transcriptase pasina RNaseH chiitiko (RNaseH-):

RNase inhibitors inowanzowedzerwa kudzoreredza maitiro ekunyora kuti awedzere kureba uye goho re cDNA synthesis.RNase inhibitor inowedzerwa mune yekutanga chain synthesis reaction muhupo hwemabuffers uye ekudzikisa maajenti akadai seDTT nekuti pre-cDNA synthesis process inobvisa inhibitor, nekudaro ichiburitsa RNase yakasungwa inosvibisa RNA.Protein RNase inhibitor inongodzivirira kuparara kweRNA neRNase A, B, C, uye haidziviriri RNase paganda, saka kuchenjerera kunofanira kutorwa kusaunza RNases kubva paminwe pasinei nekushandiswa kweizvi inhibitors.

Reverse transcriptase inokonzeresa shanduko yeRNA kuita cDNA.Ose ari maviri M-MLV uye AMV ane endogenous RNaseH chiitiko mukuwedzera kune yavo yega polymerase chiitiko.RNaseH chiitiko chinokwikwidza nepolymerase chiitiko cheheterozygous tambo dzakaumbwa pakati peRNA templates neDNA primers kana cDNA yekuwedzera tambo, uye inosvibisa RNA: RNA tambo muDNA complexes.RNA matemplate akaderedzwa neRNaseH chiitiko haachakwanise kushandiswa seanoshanda substrates yecDNA synthesis, kuderedza goho uye kureba kwecDNA synthesis.Saka kubvisa kana kudzikisira zvakanyanya RNaseH chiitiko chereverse transcriptase zvingabatsira zvikuru.

SuperScriptⅡ reverse transcriptase, MMLV reverse transcriptase yeRNaseH- uye thermoScript reverse transcriptase, AMV yeRNaseH- yakapa cDNA yakareba yakazara kupfuura MMLV neAMV.RT-PCR senitivity inokanganiswa nehuwandu hwecDNA yakagadzirwa.ThermoScript inonyanya kunzwa kupfuura AMV.Saizi yezvigadzirwa zveRT-PCR inoganhurirwa nekugona reverse transcriptase kugadzira cDNA, kunyanya kana uchigadzira maCdna akakura.Kuenzaniswa neMMLV, SuperScripⅡ yakawedzera zvakanyanya goho rezvigadzirwa zveRT-PCR refu.RNaseH-'s reverse transcriptase inowedzerawo kugadzikana kwekupisa, saka maitiro anogona kuitwa patembiricha yakakwira kupfuura yakajairwa ye37-42 ℃.Pasi pemamiriro akakurudzirwa ekugadzirisa, oligo (dT) primers uye 10μCi [alpha-p] dCTP yakashandiswa.Kugadzirwa kwese kweketani yekutanga kwakaverengerwa uchishandisa nzira yeTCA yekunaya.Hurefu hwakazara cDNA yakaongororwa pachishandiswa saizi-yakarongedzwa kubviswa tambo uye kuverenga mualkaline agarose gel.

3. Wedzera tembiricha yekuchengetedza kupisa kwereverse transcription:

Yakakwira kubata tembiricha inobatsira kuvhura yechipiri chimiro cheRNA uye kuwedzera goho rekuita.Kune akawanda matemplate eRNA, kubata iyo RNA uye primer pa65 ° C isina buffer kana munyu uye wozoitonhodza nekukurumidza paaizi inobvisa akawanda echipiri zvimiro uye inobvumira maprimers kusunga.Nekudaro, mamwe matemplate achine yechipiri chimiro, kunyangwe mushure mekupisa kwekupisa.Kukwidziridzwa kweaya matemplate akaoma kunogona kuitwa uchishandisa ThermoScript reverse transcriptase uye nekuisa reverse transcriptase reaction pane tembiricha yepamusoro kuti iwedzere kukwidziridzwa.Kubata kwepamusoro tembiricha kunogonawo kuwedzera hunhu, kunyanya kana cDNA synthesis ichiitwa pachishandiswa gene-specific primers (GSPS) (ona Chitsauko 3).Kana ukashandisa GSP, ita shuwa kuti kukosha kweTm kweprimer kwakafanana neinotarisirwa kubata tembiricha.Usashandise oligo(dT) uye zvisina tsarukano primers pamusoro pe60 ℃.Random primers inoda kubatwa pa25 ℃ kwemaminetsi gumi isati yawedzera kusvika 60 ℃.Pamusoro pekushandisa yakakwira reverse transcript tembiricha, humbowo hunogona kuvandudzwa nekuendesa zvakananga RNA/ primer musanganiswa kubva ku65 ℃ denaturing tembiricha kuenda kune reverse transcript inobata tembiricha uye nekuwedzera preheated 2 × reaction musanganiswa (cDNA thermal initiation synthesis).Iyi nzira inobatsira kudzivirira intermolecular base pairing inoitika pakudzika kwakadzika.Kushandisa PCR chiridzwa kunorerutsa akawanda tembiricha switch inodiwa kuRT-PCR.

Tth kupisa kwakagadzikana polymerase inoshanda seDNA polymerase pamberi peMg2+ uye RNA polymerase pamberi peMn2+.Inogona kuchengetedza kupisa kusvika ku65 ℃.Zvisinei, kuvapo kweMn2 + panguva yePCR kunoderedza kuvimbika, izvo zvinoita kuti Tth polymerase isanyanya kukodzera kukwidziridzwa kwepamusoro, zvakadai se cDNA cloning.Pamusoro pezvo, Tth haina kunyatso shanda pareverse transcription, iyo inoderedza kunzwisiswa, uye sezvo enzyme imwe chete inogona kuita reverse transcription uye PCR, kudzora maitiro pasina reverse transcription haigone kushandiswa kusiyanisa amplified zvigadzirwa zve cDNA kubva kune zvakasvibiswa genomic DNA.

4. Wedzerai inosimudzira reverse transcript:

Kuwedzerwa kwezvinowedzera, kusanganisira glycerin uye DMSO, kune yekutanga chain synthesis reaction inogona kuderedza kugadzikana kweiyo nucleic acid kaviri strand uye kusunungura RNA yechipiri chimiro.Kusvika ku20% glycerin kana 10% DMSO inogona kuwedzerwa pasina kukanganisa basa reSuperScriptⅡ kana MMLV.AMV inogonawo kushivirira kusvika ku20% glycerol pasina kuderedza basa.Kuti uwedzere kunzwisiswa kweRT-PCR muSuperScriptⅡ reverse transcription reaction, 10% glycerol inogona kuwedzerwa uye insulated pa45 ℃.Kana 1/10 ye retrotranscript-reaction chigadzirwa ichiwedzerwa kuPCR, kusanganiswa kweglycerol mukugadzirisa kwekusimudzira ndeye 0.4%, iyo isina kukwana kuvharidzira PCR.

5. RNaseH kugadzirisa:

Sensitivity inogona kuvandudzwa nekurapa cDNA synthesis reactions neRNaseH pamberi pePCR.Kune mamwe matemplate, zvinofungidzirwa kuti iyo RNA mu cDNA synthesis reaction inodzivirira kusungirirwa kwezvigadzirwa zvakasimudzwa, mune iyo nyaya kurapwa kweRNaseH kunogona kuwedzera kunzwa.Kazhinji, kurapwa kweRNaseH kunodiwa pakukwidziridzwa kweiyo yakareba-yakareba cDNA tarisiro yetemplate, senge tuberous scheresisⅡ ine kopi yakaderera.Kune iyi yakaoma template, RNaseH yakasimudzira chiratidzo chakagadzirwa neiyo cDNA yakagadzirwa neSuperScriptⅡ kana AMV.Kune akawanda RT-PCR maitiro, iyo RNaseH kurapwa ndeye sarudzo nekuti iyo 95 ℃ insulated PCR denaturation danho rinowanzo hydrolyze iyo RNA kubva kuRNA: DNA yakaoma.

6. Nzira dzakavandudzwa dzekuona zvidiki zveRNA:

RT-PCR inonyanya kunetsa kana zvishoma chete zveRNA zviripo.Kuwedzerwa kweglycogen semutakuri panguva yekuparadzaniswa kweRNA kunobatsira kuwedzera goho rezviduku zviduku.Iyo RNase-isina glycogen inogona kuwedzerwa panguva imwe chete seTrizol.Glycogen inonyungudika mumvura uye inogona kuramba iri muchikamu chemvura neRNA kubatsira mukunaya kunotevera.Iyo yakakurudzirwa kuunganidzwa kweRNase-isina glycogen ndeye 250μg/ml yemasamples asingasviki 50mg yematishu kana 106 masero akagadzirwa.

Kuwedzerwa kweacetylated BSA kudzoreredza maitiro ekunyora uchishandisa SuperScriptⅡ kunogona kuwedzera kunzwisiswa, uye kune madiki eRNA, kuderedza huwandu hweSuperScriptⅡ uye kuwedzera makumi mana mayunitsi eRnaseOut nuclease inhibitor inogona kuvandudza mwero wekuona.Kana glycogen ikashandiswa mukuparadzaniswa kweRNA, kuwedzera kweBSA kana RNase inhibitors kudzoreredza maitiro ekunyora uchishandisa SuperScriptⅡ ichiri kukurudzirwa.

Ⅱ. Wedzera iyo chaiyo yeRT-PCR

1. cNDA synthesis:

Nzira nhatu dzakasiyana dzinogona kushandiswa kutanga yekutanga strand cDNA synthesis, uye hukama hweimwe nzira hunokanganisa huwandu uye rudzi rwe cDNA yakagadzirwa.

Random primer nzira ndiyo ishoma chaiyo yenzira nhatu idzi.Maprimers anonamirwa munzvimbo dzakawanda mukati mese chinyorwa kuti abudise pfupi, chidimbu kureba cDNA.Iyi nzira inowanzoshandiswa kuwana 5 ′ materminal sequences uye cDNA kubva kuRNA templates ine yechipiri dhizaini matunhu kana nekumisa masayiti ayo reverse transcriptase haigone kudzokorora.Kuti uwane iyo yakareba cDNA, chiyero cheprimers kuRNA mune yega yega sampu yeRNA inoda kutariswa zvine simba.Iyo yekutanga kuunganidzwa kwezvisina kurongeka primers inotangira pa50 kusvika 250ng pa20μl reaction system.Nekuti iyo cDNA yakasanganiswa kubva kuzere RNA uchishandisa zvisina kujairika primers inonyanya ribosomal RNA, poly(A) + RNA inowanzo sarudzwa setemplate.

Oligo (dT) kutangwa kunonyanya kujeka kupfuura kungotanga.Inosanganiswa nemuswe wepoly(A) unowanikwa kumagumo e3′ yemRNA mumaseru mazhinji eukaryotic.Nekuti poly(A) + RNA ingangoita 1% kusvika 2% yeRNA yakazara, huwandu uye kuoma kwecDNA kudiki pane kana zvakangoitika zvekutanga zvakashandiswa.Nekuda kwehukuru hwayo hwepamusoro, oligo(dT) kazhinji haidi optimization yeRNA kune primer ratio uye poly(A)+ kusarudzwa.Inokurudzirwa kushandisa 0.5μg oligo(dT) pa20μl reaction system.oligo(dT)12-18 inokodzera akawanda RT-PCR.ThermoScript RT-PCR System inopa oligo(dT)20 nekuda kwekugadzikana kwayo kwemafuta uye inokodzera kudziya kwepamusoro.

Gene-specific primers (GSP) ndiwo akanakisa ekutanga eiyo reverse transcription nhanho.GSP is an antisense oligonucleoside iyo inogona kunyanya kusanganisa neRNA yekuenda sequences, pane anneal ese maRna senge asina kujairika primers kana oligo(dT).Mitemo inoshandiswa kugadzira PCR primers inoshandawo kune dhizaini reverse transcription reaction GSP.GSP inogona kuita kutevedzana kwakafanana neyekukwidziridza primer yakanyudzwa pakupera kwemRNA3′, kana GSP inogona kugadzirwa kuti inyudzwe pasi perwizi nereverse amplification primer.Kune zvimwe zvinhu zvakakwidziridzwa, zvinodikanwa kugadzira inodarika imwe antisense primer yekubudirira RT-PCR nekuti yechipiri chimiro chechinangwa RNA chinogona kudzivirira primer kubva pakusunga.Zvinokurudzirwa kushandisa 1pmol antisense GSP mune yekutanga chain synthesis reaction system ye20μl.

2. Wedzera tembiricha yekuchengetedza kupisa kwekureverse transcription:

Kuti utore mukana wakazara weGSP chaiyo, reverse transcriptase ine kugadzikana kwemafuta kwakanyanya kunofanirwa kushandiswa.Heat-stable reverse transcriptase inogona kuiswa insulated pane tembiricha yepamusoro kuti iwedzere kuita kusimba.Semuyenzaniso, kana GSP ikavharirwa pa55°C, zvino kujeka kweGSP hakushandiswe zvizere kana reverse transcription ikaitwa pa37°C nekudzika kwakadzika uchishandisa AMV kana M-MLV.Nekudaro, SuperScripⅡ neThermoScript inogona kuita pa50 ℃ kana kupfuura, iyo inobvisa zvigadzirwa zvisina kujeka zvinogadzirwa pakudziya kwakadzika.Nekunyanya kujeka, iyo RNA / primer musanganiswa inogona kutamiswa yakananga kubva ku65 ℃ denaturation tembiricha kuenda kune reverse transcription inobata tembiricha pamwe nekuwedzera kwe preheated 2 x reaction musanganiswa (thermal initiation ye cDNA synthesis).Izvi zvinobatsira kudzivirira base pairing pakati pemamorekuru patembiricha yakaderera.Kushandisa PCR chiridzwa kunorerutsa akawanda tembiricha shanduko inodiwa kuRT-PCR.

3. Deredza genomic DNA kusvibiswa:

Imwe inogona kuoma neRT-PCR ndeyekuti RNA inosvibisa genomic DNA.Kushandiswa kwenzira dziri nani dzekuparadzanisa RNA, dzakadai seTrizol Reagent, inoderedza kusvibiswa kweDNA muRNA gadziriro.Kudzivirira zvigadzirwa zvinogadzirwa kubva kugenomic DNA, iyo RNA inogona kurapwa neamplification giredhi DnasⅠ kubvisa DNA yakasvibiswa isati yadzoreredza kunyorwa.Iwo masampula akachengetwa pa65 ℃ mu2.0mM EDTA kwemaminetsi gumi kumisa DNaseⅠ digestion.EDTA chelates magnesium ions kudzivirira iyo magnesium ion inotsamira RNA hydrolysis inoitika pakupisa kwakanyanya.

Kuitira kupatsanura amplified cDNA kubva kune genome DNA amplification chigadzirwa, maprimers ayo anneal akaparadzana neakaparadzaniswa exon anogona kugadzirwa.PCR zvigadzirwa zvakatorwa kubva kucDNA zvichave zvipfupi pane izvo zvakatorwa kubva kune yakasvibiswa genomic DNA.Chiyedzo chinodzorwa chisina reverse transcription chinoitwawo pane yega yega RNA template kuona kana chidimbu chakapihwa chinobva kugenomic DNA kana cDNA.Zvigadzirwa zvePCR zvakawanikwa pakashaikwa reverse transcription inotorwa kubva kugenome.

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