Kit ɗin FastKing RT (Tare da gDNase)

R-A-1 RNA ya lalace

——Ka tsarkake RNA mai inganci ba tare da gurɓatawa ba. Kayan da aka samo RNA daga ciki yakamata ya zama sabo gwargwado don hana ƙasƙantar da RNA. Yi nazarin amincin RNA akan gel ɗin da ba a so kafin amsawar RT. Bayan hakar RNA, yakamata a adana shi a cikin formamide 100%. Idan ana amfani da mai hana RNase, zafin zafin ya kamata ya kasance <45 ° C, kuma pH ya zama ƙasa da 8.0, in ba haka ba mai hanawa zai saki duk RNase daure. Haka kuma, yakamata a ƙara mai hana RNase a cikin mafita mai ɗauke da ≥ 0.8 mM DTT.

R-A-2 RNA ya ƙunshi masu hana haɓakar fassarar fassarar

—— Masu hana rikodin juyawa sun haɗa da SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, formamide, gishiri guanidine, da sauransu Haɗa RNA mai sarrafawa tare da samfurin, kuma kwatanta ƙimar tare da amsawar RNA mai sarrafawa don bincika ko akwai mai hanawa. Wanke hazo RNA tare da ethanol 70% (v/v) don cire masu hanawa.

A.

——Ka ƙayyade cewa zafin zafin zafin yana da dacewa da firam ɗin da aka yi amfani da su a gwajin. Don hexamers bazuwar, ana ba da shawarar kiyaye zafin jiki a 25 ° C na mintina 10 kafin a kai zafin zafin. Don takamaiman firam ɗin (GSP), gwada wasu GSP, ko canzawa zuwa oligo (dT) ko hexamer bazuwar.

A-4 Ƙananan adadin fara RNA

—— Ƙara yawan RNA. Don samfuran RNA ƙasa da 50 ng, ana iya amfani da 0.1 μg zuwa 0.5 μg acetyl BSA a farkon ƙirar cDNA

A-5 Ba a bayyana jerin maƙasudin a cikin kyallen da aka bincika ba.

——Ka gwada sauran kyallen takarda.

Amsar A-6 PCR ta gaza

—— Don matakai biyu na RT-PCR, samfurin cDNA a cikin matakin PCR ba zai iya wuce 1/5 na ƙimar amsawar ba.

A-1 Ƙaramin takamaiman firam ɗin da samfura

—— Ƙarshen 3'-firam ɗin bai kamata ya ƙunshi 2-3 dG ko dC ba. Yi amfani da takamaiman takamaiman Gene a cikin ƙirar ƙira ta farko maimakon firam ɗin bazuwar ko oligo (dT). Yi amfani da yawan zafin jiki na ƙonewa a cikin 'yan kaɗan na farko, sannan ƙananan zafin zafin. Yi amfani da farawar Taq DNA polymerase don PCR don haɓaka takamaiman halayen.

A-2 Zane mara kyau na takamaiman takamaiman halittu

——Ku bi ƙa'idodi iri ɗaya don ƙirar ƙirar share fage.

R-A-3 RNA ya gurɓata tare da kwayar halittar DNA

——Ka kula da RNA tare da DNase-PCR-matakin PCR.

A-4 Samar da dimer primer

——Zaɓi firam ɗin ba tare da jerin abubuwa masu dacewa ba a ƙarshen 3 '.

A-5 Tsayin Mg²⁺ maida hankali

——Ya inganta Mg²⁺ maida hankali ga kowane samfuri da haɗin fitila

A-6 An gurbata shi da DNA na ƙasashen waje

—— Yi amfani da nasihohi masu tsayayya da aerosol da enzymes na UDG.

A-1 Abubuwan da ke cikin samfurin ƙirar farko sun yi yawa

—— Rage adadin samfurin farko a cikin matakin amsawar PCR na al'ada.

A-2 Ya yi yawa sosai a matakin PCR

—— Rage shigarwar farko.

A-3 Hanyoyi masu yawa

——Ya haɓaka yanayin halayen PCR kuma rage lambar sake zagayowar PCR.

A-4 Ƙananan zafin zafin ƙonawa

—— Ƙara zafin zazzabi don hana ƙaddamarwa da haɓakawa ba takamaiman ba.

A-5 Ƙarin ƙayyadaddun ofan gutsuttsuran oligonucleotide wanda DNase ƙasƙantar da DNA ya haifar ——A fitar da RNA mai inganci don hana gurɓacewar DNA.

RT-PCR shine don jujjuya RNA cikin cDNA, sannan amfani da cDNA da aka sake rubutawa azaman samfuri don ɗaukar PCR don haɓaka guntun da aka nufa. Zaɓi ko dai bazuwar fitila, Oligo dT da takamaiman firam ɗin gwargwadon takamaiman yanayin gwajin. Ana iya amfani da duk firam ɗin da ke sama don gajeriyar sel eukaryotic mRNA ba tare da tsarin gashi ba.

Random primer: Ya dace da dogon RNA tare da tsarin gashin gashi, kazalika da kowane nau'in RNA kamar rRNA, mRNA, tRNA, da sauransu Ana amfani da su musamman don amsawar RT-PCR na samfuri ɗaya.

Oligo dT: Ya dace da RNA tare da wutsiyar PolyA (RNA prokaryotic, eukaryotic Oligo dT rRNA da tRNA ba su da wutsiyoyin PolyA). Saboda Oligo dT an ɗaure shi zuwa wutsiyar PolyA, ana buƙatar ingancin samfuran RNA ya zama mai girma, har ma da ƙarancin lalacewar zai rage ƙimar cikakken cDNA.

Alamar takamaimai ta Gene: Cikakken jerin samfuri, wanda ya dace da yanayin da aka san jerin maƙasudin.

Akwai hanyoyi biyu:

1. Hanyar tunani ta cikin gida: A ka'idar, cDNA gutsutsuren DNA ne na tsawon tsayi daban -daban, don haka sakamakon electrophoresis shine shafa. Idan yawan RNA ya yi ƙasa, babu samfur da zai nuna a cikin electrophoresis, amma wannan ba yana nufin babu samfur da PCR zai haɓaka ba. Gabaɗaya, ana iya amfani da bayanin ciki don gano cDNA. Idan bayanin cikin gida yana da sakamako, ana iya tabbatar da ingancin cDNA a zahiri (a cikin 'yan lokuta, idan gutsattsarin jigon da aka yi niyya ya yi tsayi sosai, ana iya samun banbanci).

2. Idan akwai sanannen ƙwayar halittar da wannan samfur ya faɗaɗa, za a iya tabbatar da shi ta farkon sinadarin. Ofaukaka bayanin tunani na ciki ba lallai yana nufin cewa babu matsala tare da cDNA ba. Saboda bayanin ciki yana da yawa a cikin cDNA, yana da sauƙin haɓakawa. Idan cDNA an ƙasƙantar da shi saboda dalilai daban -daban, daga hangen nesa, sakamakon PCR na ƙananan ƙwayoyin da aka yi niyya za su yi tasiri sosai. Duk da yake tunani na ciki yana da yawa a yalwace, ƙila ba za a shafa ƙarar ba.

An ƙasƙantar da RNA. Gano mutunci da tsarkake RNA

Abubuwan RNA na nau'ikan daban -daban na iya zama daban, amma gabaɗaya, jimlar RNA ɗin da aka fitar yakamata ya ƙunshi madaidaitan 28S da 18S a cikin electrophoresis na gel, kuma hasken tsohon ƙungiya ya kamata ya ninka na ƙarshen. Bandungiyar 5S tana nuna cewa RNA ta ƙasƙanci, kuma hasken ta yayi daidai da matakin ƙasƙanci. Nasarar fadada isasshen bayanin ciki baya nufin cewa babu matsala tare da RNA, saboda bayanin ciki yana da yawa, ana iya haɓaka RNA muddin lalacewar ba ta da ƙarfi. OD₂₆₀/OD₂₈₀rabo na RNA mai tsabta wanda aka auna ta spectrophotometer yakamata ya kasance tsakanin 1.9 da 2.1. Ƙananan ƙarancin ƙazantar furotin a cikin RNA zai rage rabo. Muddin ƙimar ba ta yi ƙasa sosai ba, RT ba za ta shafa ba. Abinda yafi mahimmanci ga RT shine amincin RNA.

Tsawaita jigon tunani na ciki na iya nuna kawai cewa RT ta yi nasara, amma ba lallai ne ya danganta da ingancin igiyar cDNA ba. Saboda gutsattsarin tunani na ciki gabaɗaya ƙanana ne kuma babba a cikin magana, sun fi sauƙi don samun nasara a jujjuyawar juyawa. Duk da haka, girman da kuma bayyana jigidar da aka yi niyya ta bambanta daga gene zuwa gene. Ba za a iya tantance ingancin cDNA kawai ta hanyar tunani na ciki ba musamman don gutsutsuren da aka yi niyya fiye da 2 kb.

Wasu samfuran suna da sifofi masu rikitarwa na biyu, ko suna da wadataccen abun ciki na GC, ko suna da daraja tare da ƙarancin yalwa. A cikin waɗannan lamuran, yakamata a zaɓi transcriptase daidai gwargwadon girman gutsattsarin manufa da samfurin. Don samfuran RNA tare da babban abun ciki na GC da hadaddun tsarin sakandare, yana da wahala a buɗe tsarin sakandare a ƙarancin zafin jiki, ko tare da juzu'in juzu'in gama gari. Don waɗannan samfuran, ana iya zaɓar Quant Reverse Transcriptase, tunda aikin jujjuyawar jujjuyawar sa a bayyane ya fi na M-MLV jerin juzu'in juzu'i, wanda zai iya jujjuya samfuran RNA daban-daban yadda yakamata kuma ya rubuta RNA zuwa cDNA tungar farko zuwa matsakaicin matsayi. Lokacin amfani da kit ɗin transcriptase gaba ɗaya, tsarin 20 μl zai iya jujjuya rubutaccen 1 μg na jimlar RNA. Da fatan za a kula da iyakar ƙarfin RT na kit. Idan an ƙara samfuri fiye da kima, kwafin juzu'in zai fifita RNA da yawa. Sabili da haka, yana da kyau kada ku wuce matsakaicin ƙarfin tsarin.

A-1 Tabbatar idan RNA ta lalace sosai kuma idan RT tayi nasara

Gabaɗaya, dalilin gazawar ƙaramin tunani na ciki galibi yana haifar da lalacewar RNA mai mahimmanci. Wani mawuyacin dalili shine gazawar kwafi na juyawa. Ba za a iya amfani da nassoshi na cikin gida azaman ma'auni don yin hukunci da ingancin layin cDNA guda ɗaya ba, amma ana iya amfani da shi azaman ma'auni don yin hukunci ko jujjuyawar nasara ta yi nasara idan babu matsalar ingancin RNA. Abu mafi mahimmanci a cikin tsarin jujjuyawar juzu'i shine kula da zazzabi mai ɗorewa da tsarin amsawa akai -akai domin inganta ingancin aiki.

A-2 ƙayyade ko firam ɗin don haɓaka ƙwayoyin tunani na ciki amintacce ne kuma idan akwai matsaloli tare da reagents da ake amfani da su a PCR.

Don ƙididdige dangi, dole ne a ƙididdige RNA kafin jujjuyawar juzu'i, wanda kuma ake buƙata a cikin kayan jujjuyawar juzu'i, alal misali, ƙididdige shigarwar RNA azaman 1 μg. Tunda cDNA mai jujjuyawar bayanai shine cakuda mai hade, gami da RNA, oligo dT, enzyme, dNTP, har ma da ragowar DNA, za a sami karkacewa, don haka ba zai yiwu a ƙididdige cDNA daidai ba. Don haka, ƙididdigar RNA ya zama dole. La'akari da ingancin jujjuyawar juzu'i iri ɗaya ne tsakanin samfura daban -daban, adadin cDNA da aka samu ya zama iri ɗaya, kuma ƙididdigar ƙididdiga na iya nuna kwatancen matakan magana na kwayoyin halittu daban -daban a cikin adadin jimlar RNA. Lokacin yin PCR mai ƙima mai ƙima, ƙila ba za a buƙaci cDNA mai yawa ba bayan jujjuyawar juyi saboda ana iya yin amfani da jigon bayanin ciki.

Yana da alaƙa da kwayoyin halitta, kuma jujjuyawar juzu'in dogon guntu ba zai yiwu ba ga yawancin kwayoyin halitta. Da fari, ingancin jujjuyawar juyawa ya yi ƙasa da na PCR. Abu na biyu, yankin GC mai wadata da tsarin sakandare na ƙwayoyin halittu da yawa suna taƙaita duka juzu'in juyi da PCR. A ƙarshe, aminci da haɓaka ƙarfin PCR suna da wuyar tabbatarwa a lokaci guda. A cikin tsarin jujjuyawar juyawa, babu wanda zai iya ba da tabbacin samun dogon guntu don ƙarancin kwafin ƙwayoyin cuta, musamman ta amfani da oligo dT. Game da 5 'UTR tare da ƙarin GC, ya fi wahala. Sabili da haka, har yanzu hanya ce mai dacewa don jujjuya kwafi tare da bazuwar bazuwar, nemo wuraren rarrabuwar dabi'a a cikin gutsattsarin manufa, ƙara haɓaka ta sashi, sannan aiwatar da ƙuntatawa da haɗawa. Gabaɗaya, yana da wahalar haɓaka gutsutsuren da ya fi 2 kb kai tsaye, amma ba koyaushe ba zai yiwu a samu: 1. Da farko, tabbatar da amincin RNA/mRNA, kuma an fi son haɓakar TRIZOL. 2.M-MLV RT-PCR kit za'a iya amfani dashi kai tsaye. Ƙara lokacin ƙarawa da haɓaka lambar sake zagayowar a cikin tsarin haɓakawa da kyau. A madadin haka, ana iya amfani da PCR da aka saka, ko aiwatar da halayen guda ɗaya ko biyu da farko tare da tsawaita ƙaddamarwa da lokacin haɓaka kafin haɓaka PCR na yau da kullun, wanda na iya taimakawa wajen fadada gutsuttsura. Kula da amincin polymerase. 3.Long Taq ana iya amfani dashi a cikin PCR don samun kyakkyawan sakamako. 4.Domin aikace -aikacen bayyana furotin, yakamata a yi amfani da polymerase mai aminci.

Akwai nau'ikan juzu'in juzu'i biyu waɗanda TIANGEN ke bayarwa: Quant/King RTase da TIANScript M-MLV. Babban bambanci tsakanin su shine adadin shigar samfura. Quant shine transcriptase na musamman, wanda ya bambanta da M-MLV da aka saba amfani da shi wanda aka samo daga cutar sankarar sankara ta Moloney murine. Quant shine sabon ingantaccen ingantaccen transcriptase wanda aka sake bayyanawa ta hanyar injiniyan Escherichia coli. Quant ya dace don haɓaka 50 ng-2 μg na RNA tare da babban aikin juzu'in juyi da yawan amfanin ƙasa. Idan aka kwatanta da MMLV ko AMV na yau da kullun, babban halayyar Quant ita ce tana da alaƙa mai ƙarfi tare da samfuran RNA kuma tana iya jujjuya samfuran rikodin rikodin ba tare da ƙirar zazzabi mai zafi ba. Don samfura tare da babban abun ciki na GC, ingantaccen juzu'in ya fi girma. Koyaya, wannan juzu'in juzu'in yana da aikin RNase H, wanda zai iya shafar tsawon samfurin cDNA (wanda ya dace da <4.5 kb samfura). Don fassarar jujjuyawar al'ada, an ba da shawarar TIANScript MMLV transcriptase na baya. Wannan RTase wani enzyme ne wanda aka gyara tare da raunin RNase H mai rauni sosai, wanda ya dace da haɗin cDNA mai tsawo (> 5 kb).

An kammala fassarar juzu'i ɗaya da haɓaka PCR a cikin bututu ɗaya ba tare da buɗe murfin bututu tsakanin haɗin cDNA da haɓakawa ba, wanda ke taimakawa rage gurɓatawa. Tunda duk samfuran cDNA da aka samu ana amfani da su don haɓakawa, ƙwarewar ta fi girma, tare da mafi ƙarancin 0.01 pg na jimlar RNA. Don nasarar RTPCR na mataki ɗaya, galibi ana amfani da firam ɗin musamman don fara kira cDNA. Hanyar matakai biyu, wato juzu'in juyawa da haɓaka PCR ana aiwatar da shi cikin matakai biyu. Da farko ana jujjuya juzu'in juzu'i daga samfuran RNA don samun cDNA, kuma cDNA da aka samu yana fuskantar halayen PCR ɗaya ko fiye. Hanyar matakai biyu na iya amfani da oligo (dT) ko firam ɗin bazuwar don jagorantar kira na farkon cDNA, kuma yana iya jujjuya rubuta duk bayanan mRNA daga takamaiman samfurin.