Gwajin RT-qPCR ya haɗa da cirewar RNA da ƙimar inganci, jujjuya rubutu da qPCR matakai uku, kowane mataki yana da matakan tsaro da yawa, zamu gabatar da dalla-dalla a ƙasa.

Ⅰ.kimanta ingancin RNA

A cikin gwajin RT-qPCR, bayan an gama cirewar RNA, ana buƙatar kimanta ingancin RNA, kuma gwajin na gaba za a iya aiwatar da shi bayan ya cancanta.Hanyoyin kimantawa sun haɗa da spectrophotometer, Agilent gel electrophoresis, Agilent 2100 bincike, daga cikinsu mafi yawan amfani da spectrophotometer da agarose gel electrophoresis hanyar ganowa.Ya kamata a lura cewa waɗannan hanyoyi guda biyu suna buƙatar amfani da su tare don kammala ganowa da kuma nazarin tattarawar RNA, tsabta da mutunci, don tabbatar da ingancin RNA.

Kayan Keɓewar RNA mai alaƙa: 

Salon Warewa Kit ɗin RNA

Za'a iya samun jimlar RNA mai tsabta da inganci daga ƙwayoyin al'ada daban-daban a cikin mintuna 11.

Jimlar Dabbobin Keɓewar RNA

Cikin sauri da inganci cire tsafta mai inganci da jimillar RNA daga kyallen dabbobi daban-daban.

Spectrophotometer:

Ana amfani da spectrophotometer musamman don tantance taro da tsabtar RNA, amma ba zai iya gano amincin RNA da ragowar kwayoyin halitta ba.Daga cikin su, A260/280 da A260/230 mahimman sigogi ne don gano tsaftar RNA, kuma ana iya gano tsaftar RNA bisa ga jujjuyawar kimarsu:

1. 1.9 2.1, yana nuna yiwuwar ɓarna na RNA, wanda za'a iya tabbatar da shi ta hanyar agarose gel electrophoresis.

2. 2.0

Agarose gel electrophoresis:

Agarose gel electrophoresis assay na iya yin nazarin amincin RNA, kwayoyin halitta da ragowar furotin, amma ba zai iya ƙididdige yawan adadin RNA daidai ba ko gano ragowar reagents na halitta.Ɗauki samfuran eukaryotic RNA misali:

1. An sanya RNA ga agarose gel electrophoresis.Idan akwai nau'i-nau'i guda uku na 28sRNA, 18sRNA da 5.8sRNA akan taswirar gel, yana nuna cewa RNA da aka fitar ba ta nan.Idan akwai abin jan hankali, yana nuna ɓarna na RNA.

2. Idan akwai bandeji mai haske guda ɗaya tsakanin ramin manne da rukunin 28sRNA, ana iya samun ragowar DNA na genomic.

3. Idan bandeji sun bayyana a cikin rami mai manne, yana nuna cewa za'a iya samun ragowar furotin da sauran abubuwan macromolecular.

Ⅱ. Juya rubutun

Bayan an gama cirewar RNA, yana buƙatar juyawa zuwa cDNA don gwaje-gwaje na gaba, don haka matakin juyawa yana da mahimmanci.Za a gabatar da fassarar juzu'i daga zaɓin juzu'i na juzu'i da firamare:

Sake zaɓin fassarar fassarar:

Matsalolin juzu'i na yau da kullun sun haɗa da AMV RTase da MMLV RTase.RNase H na AMV RTase yana da aiki mai karfi, gajeren tsayin kira, ƙananan ƙira da kwanciyar hankali mai kyau (42 ~ 55 ℃).Ayyukan RNase H na MMLV RTase yana da rauni, tsayin kira yana da tsawo, adadin kira yana da girma, kuma kwanciyar hankali na thermal ba shi da kyau (37 ~ 42 ℃).

Saboda RNase H enzyme yana da aikin ɓata samfurin RNA, MMLV tare da raunin RNase H ya kamata a zaɓa da kyau yayin rubuta juzu'i, kuma bayan injiniyan ƙwayoyin halitta daga baya, kwanciyar hankali na zafin jiki na MMLV ya kai matsayi mai inganci.Samun ForegeneFassarar juzu'i na Foreasy (M-MLV don juyawa baya) a matsayin misali, sabon juzu'i ne da aka bayyana a cikin E. coli injiniyoyin ƙwayoyin cuta ta amfani da fasahar sake haɗuwa da kwayoyin halitta.Wani recombinant DNA polymerase wanda ke haɗa madaidaicin madaidaicin DNA daga RNA, DNA, ko RNA: DNA hybrid.Ba shi da aikin RNase H, kwanciyar hankali mai ƙarfi, ƙaƙƙarfan alaƙar RNA, da babban ganewar ganewa.

 

Fassarar juzu'i na Foreasy (M-MLV don juyawa baya)

Zaɓin na farko:

Gabaɗaya RT primers sun faɗi cikin nau'i uku: oligo dT, bazuwar ƙa'idodi, da ƙayyadaddun ƙirar halitta.Zaɓi madaidaitan madaidaitan don amfani bisa ga buƙatun gwaji daban-daban.

1. Idan samfuri na asalin eukaryotic ne kuma ana amfani da ƙarshen cDNA don haɓaka PCR na yau da kullun, ana ba da shawarar Oligo (dT);Idan gwajin na gaba an yi amfani da shi kawai don qPCR, Oligo (dT) ana ba da shawarar a haɗe shi tare da bazuwar maɓalli don inganta ingantaccen juzu'i.

2. Idan samfurin ya fito daga prokaryotes, Random Primers ko takamaiman al'amuran halitta ya kamata a zaɓi su don juyar da rubutu.

Ⅲ.qPCR

Ƙididdigar fluorescence galibi an ƙaddamar da shi daga zaɓin hanyoyin ƙididdigewa, ƙa'idodin ƙirar ƙira, zaɓin ROX, daidaitawar tsarin amsawa da saitin yanayin amsawa, da sauransu.

Zaɓin hanyoyin ƙididdigewa:

Hanyoyi masu ƙididdigewa sun kasu zuwa hanyoyin ƙididdiga na dangi da cikakkun hanyoyin ƙididdigewa.Ana iya amfani da ƙididdige ƙididdigewa don gano tasirin wasu hanyoyin jiyya akan maganganun kwayoyin halitta, gano bambancin maganganun kwayoyin halitta a lokuta daban-daban da kwatanta bambancin maganganun kwayoyin halitta a cikin kyallen takarda daban-daban.Cikakken ƙididdigewa zai iya gano adadin nucleic acid a cikin ƙwayar cuta da sauransu.Lokacin yin gwaje-gwaje, dole ne mu zaɓi hanyoyin ƙididdiga masu dacewa bisa ga namu gwaje-gwaje.

Ka'idodin ƙira na farko:

Zane na farko don qPCR yana da alaƙa kai tsaye da haɓaka haɓakawa da ƙayyadaddun samfur.Saboda haka, daidai zayyana nagartattu masu kyau shine matakin farko na qPCR mai nasara.A cikin ƙirar ƙira, ya kamata a kula da ka'idodin masu zuwa yayin saduwa da ƙa'idar ƙirar ƙirar al'ada:

1. Tsawon guntuwar da aka yi niyya ana sarrafawa tsakanin 100 da 300 bp;

2. Tsarin giciye-exon don kauce wa tasirin DNA na genomic;

3. Abubuwan da aka ƙera suna buƙatar gwadawa don haɓaka haɓakawa, kuma kawai lokacin da ƙarfin haɓaka ya kai daidaitattun (90-110%) za a iya amfani da su don gwaje-gwaje masu yawa;

4. Ana inganta maida hankali na farko tsakanin 0.1uM da 1.0uM.

ZaɓinROX:

A cikin aiwatar da ƙimar ƙididdiga, ROX na iya daidaita bambance-bambancen hanyar gani, kuskuren pipetting ko bambance-bambancen ƙarar da ke haifar da evaporation da condensation iri ɗaya, haɓaka maimaita sakamakon.Koyaya, ya kamata a lura cewa zaɓin ROX yana da alaƙa da kayan aikin.Idan kayan aikin qPCR yana da aikin gyara bambanci tsakanin ramuka ta atomatik, baya buƙatar ƙara ROX;in ba haka ba, yana buƙatar ƙara gyaran ROX.Ƙananan abokan haɗin gwiwa a cikin siyan reagents dole ne su kasance daidai da kayan aikin da aka yi amfani da su don zaɓar ROX daidai, guje wa kurakurai daga baya.

Shiri tsarin amsawa:

An fi son juzu'in martani na 20ul da 50ul.Ya kamata a kula da abubuwan da suka biyo baya lokacin da aka tsara tsarin:

1. Ana buƙatar tsarin amsawa ta hanyar samun iska a cikin benci mai tsabta mai tsabta, sabon ddH₂Ana amfani da O don kowane gwaji;

2. Kowane gwaji yana buƙatar shirya NTC don tabbatar da ko akwai gurbatawa a cikin tsarin, kuma kowane nau'i na farko yana buƙatar yin NTC lokacin shirya tsarin;

3. Don gano ko akwai ragowar gDNA a cikin samfurin RNA, ana iya shirya NRT don kowane samfurin don ganowa;

4. Lokacin shirya tsarin, ana bada shawarar yin aƙalla 3 sake maimaita fasaha don samfurin daya;

5. Lokacin da samfurin ya kasance cDNA, ana bada shawara don tsarma sau 5-10 don rage tasirin hanawa na tsarin rubutun baya akan gwajin qPCR.Yana da kyau a bincika yawan samfurin ta hanyar gradient, don haka ƙimar CT ta faɗi tsakanin 20-30;

6. Ƙayyade adadin halayen da ake buƙata, ƙara da 5-10% bisa yawan adadin halayen, kuma ƙididdige lambar ƙirar ƙarar;

7, an shirya tsarin ta amfani da ka'idar premix, haɗuwa bayan centrifugation kuma tabbatar da babu kumfa;

8, Kamar yadda zai yiwu don zaɓar kayan amfani masu goyan baya.

Kit ɗin RT-qPCR mai alaƙa