Ⅰ. Ƙara hankali na tsarin amsawa:

1. Rarrabe babban ingancin RNA:

Nasarar haɗin cDNA yana fitowa daga RNA mai inganci.RNA mai inganci yakamata ya tabbatar da aƙalla jimlar tsayi kuma baya ƙunshi masu hanawa waɗanda basu ƙunshi enzymes rikodi ba, kamar EDTA ko SDS.Ingancin RNA yana ƙayyade matsakaicin ƙimar bayanan jeri da za ku iya rubutawa zuwa cDNA.Hanyar tsarkakewa ta RNA gabaɗaya hanya ce ta amfani da isoocyanate/acidophenol.Don hana gurɓacewar RNase, RNA da aka rabu da samfurin mai arziki a cikin RNase (kamar pancreas) yana buƙatar adana formaldehyde don adana RNA mai inganci, wanda ya fi haka don adana dogon lokaci.RNA da aka ciro daga hantar bera ta ragu sosai bayan sati guda na ajiya a cikin ruwa, yayin da RNA da aka ciro daga magudanar bera ta tsaya tsayin daka bayan kwashe shekaru uku a cikin ruwa.Bugu da kari, bayanan da suka fi girma fiye da 4kb sun fi kula da gano lalacewar RNase fiye da kananan kwafi.Don ƙara kwanciyar hankali na samfurin RNA na ajiya, ana iya narkar da RNA a cikin methalmamine na ion, kuma ana adana shi -70 ° C.Thylide da ake amfani da shi don ajiye RNA ba dole ba ne ya ƙunshi wani abu dabam wanda ke ƙasƙantar da RNA.RNA, wanda aka samo daga pancreas, ana iya adana shi a cikin methalmamine na akalla shekara guda.Lokacin da kake shirye don amfani da RNA, zaka iya amfani da hanyoyi masu zuwa don haɓaka RNA: ƙara NaCl zuwa 0.2m kuma sau 4 na ƙarar ethanol, sanya dakin da zafin jiki na minti 3-5, da 10,000 × g centrifugal na minti 5.

2. Yi amfani da juzu'i ba tare da aikin RNaseH ba (RNaseH-):

Ana ƙara masu hana RNase sau da yawa don juyar da halayen rubutun don ƙara tsayi da yawan amfanin haɗin cDNA.Ana ƙara inhibitor na RNase a cikin haɗin haɗin sarkar farko a gaban masu buffers da rage wakilai kamar DTT saboda tsarin haɗin pre-cDNA yana hana mai hanawa, ta haka yana sakin RNases masu ɗaure waɗanda ke lalata RNA.Protein RNase inhibitor kawai yana hana lalacewar RNA ta hanyar RNase A, B, C, kuma baya hana RNases akan fata, don haka yakamata a kula da kar a gabatar da RNases daga yatsun hannu duk da amfani da waɗannan masu hanawa.

Reverse transcriptase yana haifar da jujjuyawar RNA zuwa cDNA.Dukansu M-MLV da AMV suna da ayyukan RNaseH na ƙarshe ban da nasu ayyukan polymerase.Ayyukan RNaseH yana gasa tare da ayyukan polymerase don nau'ikan heterozygous da aka kafa tsakanin samfuran RNA da abubuwan haɓaka DNA ko tsattsauran ra'ayi na cDNA, kuma suna ƙasƙantar da RNA: igiyoyin RNA a cikin rukunin DNA.Samfuran RNA da ayyukan RNaseH suka ƙasƙanta ba za a iya amfani da su azaman ingantattun ma'auni don haɗin cDNA ba, rage yawan amfanin ƙasa da tsayin haɗin cDNA.Don haka kawar ko rage yawan ayyukan RNaseH na juyar da rubutun zai zama babban fa'ida.

SuperScriptⅡ juzu'i na juyi, MMLV mai juye juzu'i na RNaseH- da thermoScript juzu'i na juzu'i, AMV na RNaseH- ya samar da ƙarin cikakken tsayin cDNA fiye da MMLV da AMV.Adadin cDNA da aka haɗa yana shafar hankalin RT-PCR.ThermoScript ya fi AMV kulawa sosai.Girman samfuran RT-PCR yana iyakance ta ikon jujjuya rubutun don haɗa cDNA, musamman lokacin rufe manyan Cdnas.Idan aka kwatanta da MMLV, SuperScripⅡ yana haɓaka yawan amfanin dogayen samfuran RT-PCR.RNaseH-'s reverse transcriptase shima yana ƙaruwa da kwanciyar hankali, don haka ana iya aiwatar da martanin a yanayin zafi sama da na al'ada na 37-42 ℃.A ƙarƙashin sharuɗɗan haɗakar da aka ba da shawarar, an yi amfani da madaidaicin oligo (dT) da 10μCi [alpha-p] dCTP.An ƙididdige jimlar samar da sarkar farko ta amfani da hanyar hazo TCA.An yi nazarin cDNA mai cikakken tsayi ta amfani da cire tsiri mai girma da kirga a cikin gel na agarose na alkaline.

3. Ƙara zafin adana zafi na juyar da rubutu:

Mafi girman zafin jiki yana taimakawa buɗe tsarin na biyu na RNA kuma yana ƙara yawan abin da ake samu.Don mafi yawan samfuran RNA, riƙe RNA da firamare a 65°C ba tare da buffer ko gishiri ba sannan sanyaya su da sauri akan kankara yana kawar da yawancin tsarin sakandare kuma yana ba da damar daɗaɗɗa.Koyaya, wasu samfuran har yanzu suna da tsari na biyu, koda bayan ƙarancin zafi.Ana iya haɓaka waɗannan samfura masu wahala ta amfani da ThermoScript reverse transcriptase da kuma sanya juzu'in juzu'i a yanayin zafi mafi girma don haɓaka haɓakawa.Hakanan maɗaukakin yanayin zafi na iya ƙara ƙayyadaddun ƙayyadaddun bayanai, musamman lokacin da ake yin haɗin cDNA ta amfani da ƙayyadaddun ƙayyadaddun kwayoyin halitta (GSPS) (duba Babi na 3).Idan ana amfani da GSP, tabbatar da cewa ƙimar Tm na firamare daidai yake da yawan zafin da ake sa ran.Kada a yi amfani da oligo(dT) da bazuwar firamare sama da 60 ℃.Dole ne a gudanar da abubuwan farko na bazuwar a 25 ℃ na mintuna 10 kafin ƙara zuwa 60 ℃.Baya ga yin amfani da yanayin juyi mafi girma, ana iya haɓaka ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai ta hanyar canja wurin cakuda RNA/primer kai tsaye daga madaidaicin zafin jiki na 65 ℃ zuwa jujjuyawar juzu'i mai riƙe zafin jiki da ƙara cakudawar amsawar 2 × preheated (haɗuwar ƙaddamarwar thermal cDNA).Wannan hanya tana taimakawa hana haɗin haɗin ginin tsakanin kwayoyin halitta wanda ke faruwa a ƙananan yanayin zafi.Yin amfani da kayan aikin PCR yana sauƙaƙa da yawan canjin zafin jiki da ake buƙata don RT-PCR.

Tth zafin da aka daidaita polymerase yana aiki azaman DNA polymerase a gaban Mg2+ da RNA polymerase a gaban Mn2+.Yana iya ɗaukar zafi har zuwa 65 ℃.Koyaya, kasancewar Mn2+ a lokacin PCR yana rage aminci, wanda ke sa Tth polymerase ya zama ƙasa da dacewa don haɓaka daidaitattun daidaito, kamar cDNA cloning.Bugu da kari, Tth ba shi da inganci a jujjuyawar juzu'i, wanda ke rage hankali, kuma tunda guda ɗaya enzyme na iya yin juzu'i da PCR, halayen sarrafawa ba tare da juyar da juzu'i ba ba za a iya amfani da su ba don bambanta haɓakar samfuran cDNA daga na gurɓataccen DNA.

4. Ƙarin da ke inganta rubutun baya:

Ƙarin abubuwan da aka ƙara, ciki har da glycerin da DMSO, zuwa aikin haɗakarwa na farko na iya rage kwanciyar hankali na nau'in acid nucleic kuma ya kwance tsarin na biyu na RNA.Ana iya ƙara har zuwa 20% glycerin ko 10% DMSO ba tare da shafar ayyukan SuperScriptⅡ ko MMLV ba.AMV kuma na iya jure har zuwa 20% glycerol ba tare da rage aiki ba.Don haɓaka hankalin RT-PCR a cikin SuperScriptⅡ amsawar juzu'i, ana iya ƙara 10% glycerol kuma a rufe shi a 45 ℃.Idan an ƙara 1/10 na samfurin retrotranscription-reaction zuwa PCR, ƙaddamarwar glycerol a cikin haɓakar haɓakawa shine 0.4%, wanda bai isa ya hana PCR ba.

5. sarrafa RNaseH:

Ana iya inganta hankali ta hanyar magance halayen haɗin haɗin cDNA tare da RNaseH kafin PCR.Ga wasu samfura, ana tunanin cewa RNA a cikin halayen haɗin gwiwar cDNA yana hana ɗaurin samfuran haɓakawa, wanda a cikin yanayin jiyya na RNaseH na iya haɓaka hankali.Gabaɗaya, ana buƙatar jiyya na RNaseH don haɓaka samfurin cDNA mai cikakken tsayi mai tsayi, kamar scherosis Ⅱ tuberous tare da ƙaramin kwafi.Don wannan samfuri mai wahala, RNaseH ya haɓaka siginar da cDNA ta haɗa ta SuperScriptⅡ ko AMV.Ga mafi yawan halayen RT-PCR, magani na RNaseH zaɓi ne saboda 95 ℃ keɓaɓɓen matakin denaturation na PCR yawanci yana sanya RNA daga RNA: hadaddun DNA.

6. Ingantattun hanyoyin gano ƙananan adadin RNA:

RT-PCR yana da ƙalubale musamman lokacin da ƙananan adadin RNA ke samuwa.Bugu da ƙari na glycogen a matsayin mai ɗauka yayin rabuwar RNA yana taimakawa wajen ƙara yawan ƙananan samfurori.Ana iya ƙara glycogen maras RNase a lokaci guda da Trizol.Glycogen mai narkewa ne da ruwa kuma yana iya kasancewa cikin yanayin ruwa tare da RNA don taimakawa a hazo mai zuwa.Matsakaicin shawarar glycogen maras RNase shine 250μg/ml don samfuran ƙasa da 50mg na nama ko ƙwayoyin al'ada 106.

Ƙarin BSA acetylated don juyar da halayen rubutu ta amfani da SuperScriptⅡ na iya ƙara hankali, kuma ga ƙananan adadin RNA, rage adadin SuperScriptⅡ da ƙara raka'a 40 na RnaseOut nuclease inhibitor na iya inganta matakin ganowa.Idan ana amfani da glycogen a cikin rabuwar RNA, ƙari na BSA ko RNase inhibitors don juyar da halayen rubutu ta amfani da SuperScriptⅡ har yanzu ana ba da shawarar.

Ⅱ. Ƙara takamaiman RT-PCR

1. CNDA kira:

Ana iya amfani da hanyoyi daban-daban guda uku don fara haɗin cDNA na farko, kuma ƙayyadaddun dangi na kowace hanya yana rinjayar adadin da nau'in cDNA da aka haɗa.

Hanyar farko ta bazuwar ita ce ƙayyadaddun ƙayyadaddun hanyoyin guda uku.Ana share maɓalli a wurare da yawa a duk cikin rubutun don samar da gajeren cDNA mai tsayi.Ana amfani da wannan hanyar sau da yawa don samun jeri na ƙarshe na 5′ da cDNA daga samfuran RNA tare da yankuna na biyu ko tare da wuraren ƙarewa waɗanda ke juyar da rubutun ba za su iya yin kwafi ba.Don samun mafi tsayin cDNA, ana buƙatar ƙaddara rabon firamare zuwa RNA a cikin kowane samfurin RNA a zahiri.Matsakaicin farko na firamare bazuwar jeri daga 50 zuwa 250ng a kowace 20μl dauki tsarin.Saboda cDNA da aka haɗe daga jimillar RNA ta amfani da bazuwar firamare galibi RNA ribosomal ne, ana zaɓar poly(A)+ RNA gabaɗaya azaman samfuri.

Ƙaddamarwar Oligo(dT) ta fi ƙayyadaddun ƙayyadaddun abubuwan da ba a so ba.Yana haɓaka tare da wutsiyar poly(A) da aka samo a ƙarshen 3′ na mRNA a yawancin ƙwayoyin eukaryotic.Saboda poly(A)+RNA shine kusan 1% zuwa 2% na jimillar RNA, adadin da sarkar cDNA ya yi ƙasa da idan an yi amfani da bazuwar firamare.Saboda ƙayyadaddun ƙayyadaddun sa, oligo(dT) gabaɗaya baya buƙatar haɓakawa don RNA zuwa matakin farko da zaɓin poly(A)+.Ana ba da shawarar yin amfani da 0.5μg oligo(dT) akan tsarin amsawa na 20μl.oligo(dT)12-18 ya dace da yawancin RT-PCR.ThermoScript RT-PCR System yana ba da oligo (dT) 20 saboda kyakkyawan yanayin yanayin zafi kuma ya dace da yanayin zafi mai girma.

Ƙayyadaddun ƙayyadaddun kwayoyin halitta (GSP) sune mafi kyawun ƙayyadaddun firikwensin don matakin juyar da rubutu.GSP shine oligonucleoside antisense wanda zai iya haɓaka musamman tare da jeri na RNA, maimakon anneal duk Rnas kamar bazuwar al'ada ko oligo(dT).Dokokin da aka yi amfani da su don ƙirƙira abubuwan farko na PCR suma sun shafi ƙirar juyar da martanin GSP.GSP na iya zama jeri iri ɗaya da na'urar ƙarawa da aka kunna a ƙarshen mRNA3′, ko kuma ana iya ƙirƙira GSP don shafe ƙasa tare da juzu'i na ƙarawa.Ga wasu abubuwa da aka haɓaka, ya zama dole a ƙirƙira firikwensin antisense fiye da ɗaya don cin nasara na RT-PCR saboda tsarin na biyu na RNA wanda aka yi niyya na iya hana firam ɗin ɗaure.An ba da shawarar yin amfani da 1pmol antisense GSP a cikin tsarin amsawar sarkar farko na 20μl.

2. Ƙara zafin adana zafi na juyi rubutu:

Domin samun cikakkiyar fa'ida ta takamaiman GSP, ya kamata a yi amfani da jujjuya rubutun tare da ingantaccen yanayin zafi.Za'a iya keɓance juzu'in juzu'i mai zafi a yanayin zafi mai girma don ƙara ƙarfin amsawa.Misali, idan an soke GSP a 55°C, to ba a cika amfani da takamaiman GSP ba idan aka yi juyar da rubutu a 37°C tare da ƙaramin ƙarfi ta amfani da AMV ko M-MLV.Koyaya, SuperScripⅡ da ThermoScript na iya amsawa a 50 ℃ ko sama, wanda ke kawar da takamaiman samfuran da aka samar a ƙananan yanayin zafi.Don iyakar ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, ana iya canja wurin cakudawar RNA/primer kai tsaye daga zazzabi na 65 ℃ denaturation zuwa juzu'i mai riƙe zafin jiki tare da ƙari na cakuda amsawar 2 x da aka rigaya (ƙaddamarwar thermal na haɗin cDNA).Wannan yana taimakawa hana haɗin tushe tsakanin kwayoyin halitta a ƙananan zafin jiki.Amfani da kayan aikin PCR yana sauƙaƙa yawan canjin zafin jiki da ake buƙata don RT-PCR.

3. Rage gurɓatar halittar DNA:

Matsala ɗaya mai yuwuwa tare da RT-PCR ita ce RNA ta gurɓata DNA.Amfani da ingantattun hanyoyin rabuwar RNA, irin su Trizol Reagent, yana rage gurɓatar halittar DNA a shirye-shiryen RNA.Don guje wa samfuran da aka samar daga DNA na kwayoyin halitta, ana iya bi da RNA tare da ƙara darajar DNA don cire gurɓataccen DNA kafin a juyar da rubutu.An adana samfuran a 65 ℃ a cikin 2.0mM EDTA na mintuna 10 don ƙare narkewar DNAseⅠ.EDTA chelates magnesium ions don hana magnesium ion dogara RNA hydrolysis da ke faruwa a babban yanayin zafi.

Domin raba cDNA da aka haɓaka daga samfurin ƙarawa na DNA na genome, ana iya ƙirƙira abubuwan da ke ɓoye daban tare da keɓancewar exon.Kayayyakin PCR da aka samu daga cDNA za su yi guntu fiye da waɗanda aka samu daga gurɓataccen DNA na genomic.Hakanan ana yin gwajin sarrafawa ba tare da juzu'i ba akan kowane samfuri na RNA don tantance ko guntun da aka bayar ya fito daga DNA na genomic ko cDNA.Kayayyakin PCR da aka samu in babu juyar da aka rubuta an samo su ne daga kwayoyin halitta.

Samfura mai alaƙa

RT-PCR Mai Sauƙi^ᵀᴹI (Mataki Daya)

-Kit ɗin mataki ɗaya yana ba da damar juyar da rubutu da PCR don aiwatarwa a cikin bututu ɗaya.Yana buƙatar ƙara samfuri RNA kawai, ƙayyadaddun ƙirar PCR da RNase-Free ddH₂O.

-Za a iya aiwatar da ƙididdigar ƙididdigewa na RNA cikin sauri da kuma daidai.

-Kit ɗin yana amfani da na musamman na Foregene reverse reagent reagent da Foregene HotStar Taq DNA Polymerase haɗe tare da keɓaɓɓen tsarin amsawa don haɓaka ingantaccen haɓakawa da ƙayyadaddun halayen.

-Ingantaccen tsarin amsawa yana sa abin ya sami mafi girman ganewar ganewa, ingantaccen yanayin zafi, da mafi kyawun haƙuri.

RT Easy II (Tare da GDNase) Jagora Premix Don Rubutun CDNA Na Farko Don PCR na Gaskiya Tare da GDNase

-Ingantacciyar ikon cire gDNA, wanda zai iya cire gDNA a cikin samfuri a cikin mintuna 2.

-Ingantaccen tsarin jujjuya rubutu, yana ɗaukar mintuna 15 kawai don kammala haɗin layin farko na cDNA.

-Complex samfuri: samfura tare da babban abun ciki na GC da hadaddun tsarin sakandare kuma ana iya juyawa tare da ingantaccen inganci.

-Tsarin jujjuya juzu'i mai girma, samfuran pg-matakin kuma na iya samun cDNA mai inganci.

-Tsarin rubutun baya yana da babban kwanciyar hankali na thermal, mafi kyawun zafin jiki shine 42 ℃, kuma har yanzu yana da kyakkyawan aikin juzu'i a 50 ℃.