1. Gano shan maganin RNA
Abun ciki a 280, 320, 230, da 260 nm yana wakiltar ƙimar nucleic acid, baya (maganin turbidity), maida hankali na gishiri, da kwayoyin halitta kamar furotin, bi da bi.Gabaɗaya kawai dubi OD260/OD280 (Ratio, R).Lokacin da 1.8 ~ 2.0, muna tunanin cewa za'a iya jure wa gurɓataccen furotin ko wasu kwayoyin halitta a cikin RNA, amma ya kamata a lura cewa lokacin da aka yi amfani da Tris a matsayin buffer don gano abin sha, ƙimar R na iya zama mafi girma fiye da 2 (gaba ɗaya ya zama <2.2).Lokacin da R<1.8, gurɓataccen furotin ko wasu kwayoyin halitta a cikin maganin ya fi bayyane, kuma ana iya ƙayyade makomar RNA bisa ga bukatun.Lokacin da R> 2.2, yana nufin cewa an sanya RNA ruwa zuwa cikin acid nucleic guda ɗaya.
2.Electrophoretic juna na RNA
Gabaɗaya, ana amfani da gel denaturing don RNA electrophoresis, amma idan kawai don gano ingancin RNA ne, denaturing gel ba lallai ba ne, kuma ana iya amfani da gel na agarose na yau da kullun.Manufar electrophoresis shine don gano mutuncin 28S da 18S makada da rabonsu, ko amincin smear mRNA.Gabaɗaya, idan ƙungiyoyin 28S da 18S suna da haske, bayyanannu, da kaifi (yana nufin gefuna na makada a sarari), kuma hasken 28S ya ninka fiye da ninki biyu na rukunin 18S, muna ɗaukar ingancin RNA yana da kyau.
Abubuwan da ke sama sune hanyoyin guda biyu da muke amfani da su, amma babu ɗayan waɗannan hanyoyin guda biyu da zai iya bayyana mana ko akwai ragowar RNase a cikin maganin RNA.Idan akwai ƙaramin adadin RNase a cikin maganin, yana da wahala a gare mu mu gano shi tare da hanyar da ke sama, amma yawancin halayen enzymatic na gaba ana aiwatar da su a sama da digiri 37 kuma na dogon lokaci.Ta wannan hanyar, idan akwai ƙaramin adadin RNase a cikin maganin RNA, to za a sami yanayi mai dacewa da lokacin da za su taka rawarsu a gwaje-gwajen na gaba, kuma ba shakka gwajin zai yi sanyi a wannan lokacin.A ƙasa muna gabatar da hanyar da za ta iya tabbatar da ko akwai ragowar RNase a cikin maganin RNA.
3. Gwajin adana zafi
Dangane da tattarawar samfurin, zana 1000 ng RNA guda biyu daga maganin RNA kuma ƙara shi zuwa bututun centifuge na 0.5 ml, kuma ƙara shi da pH 7.0 Tris buffer zuwa jimlar ƙarar 10 ul, sannan rufe hular bututun.Saka daya daga cikinsu a cikin ruwan wanka mai zafi a 70 ° C kuma a ci gaba da dumi tsawon sa'a 1.An adana ɗayan ɓangaren a cikin firiji -20 ° C na 1 h.Lokacin da lokaci ya ƙare, cire samfurori biyu don electrophoresis.Bayan an gama electrophoresis, kwatanta igiyoyin electrophoretic na biyun.Idan makada na biyun sun yi daidai ko kuma ba su da wani muhimmin bambanci (tabbas, maƙallan su kuma sun cika sharuɗɗan a hanya ta 2), yana nufin cewa babu sauran gurɓatawar RNase a cikin maganin RNA, kuma ingancin RNA yana da kyau sosai.Akasin haka, idan samfurin da aka girka a 70°C ya nuna ɓarna a fili, yana nuna cewa akwai gurɓatawar RNase a cikin maganin RNA.
2 Hanyoyi na gwaji da dabaru don hakar RNA
Matsalolin da muke fuskanta sau da yawa lokacin fitar da RNA sune: (1) yawan amfanin RNA yana da ƙasa;(2) RNA yana da mummunar gurɓataccen gishiri;(3) RNA yana da mummunan gurɓataccen ƙarfi mai ƙarfi;(4) samfurin lalata da sauran matsaloli
1. Yawan amfani da jimillar abubuwan cirewar RNA
Hanyar guanidine isothiocyanate da hanyar Trizol sune hanyoyin da aka fi amfani dasu don fitar da jimillar RNA daga kyallen dabba da ƙwayoyin dabba.Ya dace musamman don ƙananan samfurori da kyallen takarda waɗanda ke da wahalar cirewa musamman, kamar fitar da jimillar RNA daga fatar zomo da kayan haɗin dabba;Bugu da kari, Trizol, a matsayin janar-manufa lysis reagent, kuma za a iya amfani da hakar na shuka kyallen takarda, kwayoyin, fungi da sauran kyallen takarda.Don kyallen tsire-tsire masu ɗauke da polysaccharides da polyphenols, irin su camellia oleifera, ganyen shayi, tsaban fyade, da sauransu, ana kuma iya amfani da hanyar CTAB don fitar da RNA gaba ɗaya.
A matsayin hanya ta al'ada, hanyar ginshiƙi biyu kuma ta shahara sosai saboda aikinta na zafin jiki na yau da kullun, babu buƙatar ƙara RNase, da aminci - babu chloroform, phenols da sauran reagents na halitta don hakar.(samfuran da aka ba da shawarar )
2. Cire jimlar RNA daga kyallen dabba
(1) Yi ƙoƙarin zaɓar nama mai laushi, idan ba sabo ba (zai fi dacewa a cikin watanni uku - 80 ℃ firiji ko daskararre a cikin ruwa nitrogen. Lokacin yankan nama, kada a yanke kai tsaye a dakin da zafin jiki, tabbatar da sanya shi a kan akwatin kankara, gwada ƙoƙarin guje wa daskarewa da narke maimaitawa.
(2) Yi amfani da almakashi mai tsabta da tweezers don yanke ɗan ƙaramin nama, gwada yanke tsakiyar nama lokacin yanke samfurin, ko kuma fara yanke babban nama daga tsakiya, sannan a yanke samfurin a wuri mai sanyi.Nama da aka cire ya kamata a yanke shi sosai, sanya kayan da aka daskare a cikin bututun EP ba tare da RNase ba, ƙara lysate, nama mai shredded ya kamata a bayyana shi sosai ga lysate, kuma a shirya don homogenization.
(3) Don kyallen takarda na al'ada, zaɓi nau'ikan nau'ikan nau'ikan nau'ikan wake (30-60 MG) don daidaitawa.Idan kyallen takarda sun ƙunshi babban adadin furotin, mai, ko ƙwayoyin fibrous masu yawa kamar hanta, haɓaka daidai ko rage adadin ƙwayoyin da aka yanke (na zaɓi) Zaɓi 10 ~ 20 MG).
(4) Idan ana fitar da tsokar kifi, naman shrimp, jellyfish da sauran kyallen takarda tare da babban abun ciki na ruwa, samfurin samfurin ya kamata a ƙara da kyau (shawarar 100-200 MG).
(5) Idan yanayi ya ba da izini, ana iya fitar da naman dabba kai tsaye bayan an haɗa shi tare da homogenizer na nama mai girma, idan babu irin wannan kayan aiki.
(6) RNA da aka samu bayan cirewar ƙarshe dole ne a sanya shi a kan akwatin kankara nan da nan don rage lalacewar RNA.
3. Kwayoyin RNA na dabba
(1) Kwayoyin dakatarwa: centrifuge kai tsaye kuma watsar da matsakaici, wanke tare da PBS maras kyau don 1-2 sau, sa'an nan kuma dakatar da adadin PBS mai dacewa, sa'an nan kuma ƙara lysate don lysis.Kada a ƙara lysate kai tsaye zuwa ƙwayoyin da aka haɗe bayan zubar da ruwa gaba ɗaya.Wannan zai haifar da kunshin histone da aka saki bayan sel masu lysed a saman Layer na waje su manne da waje na sel da aka haɗe, ta haka za su iyakance hulɗar sel a cikin pellet tare da lysate., yana haifar da rashin cikar ƙwayoyin sel da rage yawan amfanin RNA.
(2) Kwayoyin da ba su da mannewa ko kuma ba su da ƙarfi: Bayan watsar da matsakaici, wanke tare da PBS sau 1-2, sa'an nan kuma ɗaukar adadin PBS da ya dace kai tsaye kuma a busa tasa al'ada tare da pipette ko bindiga don busa sel, kuma canza su zuwa sel marasa RNA.Ƙara lysate zuwa bututun EP na enzyme don hakar.
(3) Kwayoyin da ke mannewa: ana buƙatar a fara narkewa tare da trypsin, sannan a tattara su cikin tubes na EP marasa RNase, a sanya su a hankali don cire supernatant, wanke sau 1-2 tare da PBS don cire wuce haddi na trypsin, kuma a sake dawowa tare da adadin PBS da ya dace sannan a ci gaba zuwa matakin cirewa.
4. Shuka RNA hakar
Tsuntsayen tsire-tsire suna da wadataccen mahalli na phenolic, ko wadatar polysaccharides, ko sun ƙunshi wasu metabolites na sakandare da ba a tantance su ba, ko suna da babban aiki na RNase.Wadannan abubuwa ana haɗe su tare da RNA bayan sel lysis don samar da hadaddun abubuwan da ba za a iya narkewa ba ko haɓakar colloidal, waɗanda ke da wahalar cirewa.Sabili da haka, lokacin da muka cire ƙwayar shuka, muna buƙatar zaɓar kayan aiki don tsire-tsire.lysate a cikin kit zai iya magance matsalolin sauƙi na iskar shaka na polyphenols da rabuwa da mahaɗan polysaccharide da acid nucleic.
(Don polysaccharide polyphenol shuka RNA hakar, samfuran da aka ba da shawarar:
(1) Bawon, ɓangaren litattafan almara, tsaba, ganye, da dai sauransu na shuka yakamata a niƙa shi sosai a cikin turmi.A lokacin aikin niƙa, ya kamata a sake cika nitrogen na ruwa a cikin lokaci don guje wa narke samfurin.Ya kamata a ƙara samfurin ƙasa da sauri a cikin lysate kuma a girgiza don guje wa lalata RNA.
(2) Don samfurori masu wadataccen fiber kamar shinkafa da ganyen alkama, yakamata a rage yawan hakowa yadda ya kamata, in ba haka ba nika nama da lysis ba zai cika ba, yana haifar da ƙarancin amfanin RNA da aka fitar.
(3) Don kyallen takarda tare da babban abun ciki na ruwa, irin su 'ya'yan rumman, 'ya'yan itacen kankana, 'ya'yan itace peach, da dai sauransu, ya kamata a ƙara girman samfurin daidai (100-200 MG shine zaɓi).
(4) Nassoshin shuka, irin su ganyen shuka, rhizomes, 'ya'yan itace masu wuya da sauran kayan ana bada shawarar amfani da nitrogen mai ruwa sosai don turmi kayan da ke cikin turmi sosai, sannan a ci gaba zuwa matakin hakowa.Al'ada nama homogenizers na iya zama ba tasiri a homogenizing shuka kyallen takarda, kuma yawanci ba da shawarar.
5. Hattara don hakar RNA
(1) Samfurin nama ya kamata ya zama sabo sosai don gujewa daskarewa da narke maimaituwa.
(2) Nama ya kamata a nitse sosai yayin da ake hakowa, kuma kada adadin nama ya yi kadan, balle ya yi yawa.
(3) Ya kamata a ba da isasshen lokacin shiryawa bayan ƙara lysate don cikakken lyse samfurin.
(4) Lokacin amfani da hanyar Trizol don hakar, ka'idar ɗaukar supernatant bayan stratification shine "fi son inhale ƙasa da yawan shaka", kuma dole ne a cire shi zuwa tsakiyar Layer, in ba haka ba zai haifar da mummunan cutar kwayar halitta ta DNA.
(5) Lokacin wankewa, ruwan wanka ya kamata ya shiga cikin bangon bututu don tabbatar da wankewa sosai.
(6) Don hanyar cire ginshiƙi, baya ga cire ginshiƙi bayan wankewa, ginshiƙin tallan ya kamata kuma a sanya shi a cikin benci mai tsafta kuma a busa tsawon mintuna 5-10 don cika ƙawancen kwayoyin halitta zuwa bushewa.
(7) A ƙarshe na hanyar ginshiƙi, bayan ƙara ruwan DEPC, sai a sanya shi na tsawon minti 3-5, ko kuma a sanya ruwan DEPC zuwa 60 ° C a gaba don ƙara yawan amfanin ƙasa.A cikin tsattsauran ra'ayi na Trizol na gargajiya da hanyar hazo na isopropanol, RNA na ƙarshe yana narkar da shi a cikin ruwan DEPC, don haka ya kamata a ba da lokacin da ya dace don rushewa, kuma a ci gaba da busa kasan bututun centrifuge tare da tip pipette.
3 Three Dalilai da mafita don ƙarancin tattarawar RNA / ƙarancin inganci
1. Yawan amfanin ƙasa yayi ƙasa da ƙasa
Samfurin da aka fitar ya yi ƙasa da ƙasa, jimlar adadin bai isa ba, ko samfurin da aka fitar ya yi yawa kuma lysis bai cika ba;ya kamata a yi amfani da nama ko sel na ingancin da ya dace don cirewa, dole ne a yi amfani da riga-kafi na samfurin da kyau, kuma lysis ya isa.
2. Ragowar kwayoyin halitta
Lokacin cirewa ta hanyar Trizol, lokacin da aka tsotse mai girma a cikin tsakiyar Layer bayan yaduwa, za a haifar da mummunar cutar genome;ya kamata a kula sosai lokacin yin shimfiɗa don guje wa tsotsa cikin tsakiyar Layer.Idan ana amfani da hanyar shafi don hakar, za'a iya zaɓar kit mai ɗauke da DNA I don hakar.Acid nucleic acid da aka tallata akan membrane yana narkewa kai tsaye tare da DNAse I, wanda zai iya rage ragowar DNA sosai.
3. Ragewar RNA
Yana iya zama lalacewar samfurin da aka fitar da kansa, ko kuma lalacewa da aka yi a lokacin aikin hakar;gwargwadon yuwuwar, yakamata a yi amfani da sabbin samfura don hakar RNA, kuma samfuran da aka tattara a adana su a cikin firiji na ruwa ko -80°C cikin lokaci, kuma a guji maimaita daskarewa da narke.Ya kamata a yi amfani da nasihun kyauta na RNase/DNase, bututun centrifuge da sauran kayan a cikin aikin hakar RNA.Tsarin hakar ya kamata ya zama da sauri kamar yadda zai yiwu.Ya kamata a sanya RNA da aka fitar akan akwatin kankara kuma a adana shi a -80 a cikin lokaci.Idan RNA da aka cire yana buƙatar ganowa ta hanyar gel electrophoresis, yakamata a yi electrophoresis nan da nan bayan an cire shi, kuma a maye gurbin buffer electrophoresis da sabon shiri.
4. Gishiri da ragowar sauran ƙarfi
Reagents na hakar sun ƙunshi phenol da guanidine gishiri, kuma maganin wanke ya ƙunshi ethanol.A lokacin aikin hakar, lysate ba a cika shi sosai ba kuma an watsar da shi, kuma maganin wankewa bai bushe ba.Ragowar gishiri da kaushi na halitta suna da illa ga juyar da rubutun da PCR na gaba.Matsayi daban-daban na hanawa, don haka lysate nama ya kamata a cire shi gaba daya yayin aikin hakar, kuma wanke ya kamata ya isa don wanke ganuwar da ke kewaye da bututu.Bugu da ƙari, an zubar da bututu kuma an busa shi mataki ne mai mahimmanci, wanda zai kara rage ragowar kwayoyin halitta.
Don ƙarin bayani game da hakar RNA, da fatan za a bi gidan yanar gizon mu:
www.foreivd.com don ƙarin bayani.
Lokacin aikawa: Dec-01-2022
