Sanannen abu ne cewa a cikin akidar tsakiya, RNA shine matsakanci na rubutu tsakanin DNA da bayanin furotin.Idan aka kwatanta da gano DNA, gano RNA zai iya yin daidai da zahirin bayyanar kwayar halitta a cikin kwayoyin halitta.Gwaje-gwajen da suka shafi RNA sun haɗa da: qRT-PCR, RNA-Seq, da gano kwayoyin halittar fusion, da sauransu. Dangane da halayen RNA kanta (zoben sukari na RNA yana da rukunin hydroxyl kyauta guda ɗaya fiye da zoben sukari na DNA), haɗe tare da adadi mai yawa na RNases a cikin mahalli, RNA ya fi rashin kwanciyar hankali kuma ya fi sauƙi a ƙasƙanta fiye da DNA.Sharar gida, datti, idan ingancin RNA ba shi da kyau, to dole ne sakamakon gwajin ya zama mara gamsarwa, musamman bayyana a matsayin bayanan da ba daidai ba ko rashin maimaitawa.Sabili da haka, ya kamata a mai da hankali sosai ga sarrafa RNA, kuma haɗin gwiwar kula da inganci kuma ya fi mahimmanci don tabbatar da daidaito da daidaito na bayanan gwaji na gaba.

Don sarrafa ingancin RNA, akwai gabaɗaya hanyoyin da aka saba amfani da su:

• Spectrophotometry
• agarose gel electrophoresis
• Agilent Bioanalyzer
• PCR mai kyalli na ainihin lokaci
• Hanyar launi na Qubit

01 Spectrophotometry

RNA yana da haɗin haɗin gwiwa biyu kuma yana da kololuwar ɗaukar nauyi a tsawon 260nm.Dangane da dokar Lambert-Beer, za mu iya lissafin tattarawar RNA daga kololuwar sha a 260nm.Bugu da kari, za mu iya kuma lissafta tsaftar RNA bisa ga rabon 260nm, 280nm da 230nm kololuwar sha.280nm da 230nm sune kololuwar shaye-shaye na sunadaran da ƙananan ƙwayoyin cuta, bi da bi.Matsakaicin A260/A280 da A260/A230 na ƙwararriyar tsaftar RNA yakamata ya fi 2. Idan bai kai 2 ba, yana nufin cewa akwai furotin ko ƙananan ƙwayoyin cuta a cikin samfurin RNA kuma ana buƙatar sake tsarkakewa.Tushen gurbatawa zai shafi gwaje-gwaje na ƙasa, kamar hana haɓaka haɓakar halayen PCR, yana haifar da ingantattun sakamako masu ƙima.Tsaftar RNA tana da babban tasiri akan sakamako na gaba, don haka spectrophotometry gabaɗaya shine hanyar haɗin sarrafa inganci mai mahimmanci a matakin farko na gwaje-gwajen acid nucleic.

Hoto 1. Hannun RNA/DNA Absorption Spectrum

02 Agarose gel electrophoresis

Baya ga tsarki, mutuncin RNA shima yana daya daga cikin muhimman alamomin tantance ingancin RNA.Lalacewar RNA zai haifar da ɗimbin guntun guntu a cikin samfurin, don haka za a rage adadin gutsuttsuran RNA waɗanda za a iya gano su yadda ya kamata da kuma rufe su ta hanyar tsarin tunani.Ana iya bincika amincin RNA ta hanyar electrophoresis na jimlar RNA akan gel na agarose 1%.Wannan hanyar za ta iya saita gel ɗin da kanka, ko amfani da tsarin E-Gel™ da aka riga aka keɓance don gwada gaskiya.Fiye da 80% na jimillar RNA ribosomal RNA ne, mafi yawansu sun ƙunshi 28S da 18S rRNA (a cikin tsarin dabbobi masu shayarwa).Kyakkyawan ingancin RNA zai nuna sanduna masu haske guda biyu masu haske, waɗanda sune sanduna masu haske na 28S da 18S, bi da bi, a 5 Kb da 2 Kb, kuma rabon zai kasance yana kusa da 2:1.Idan yana cikin yanayi mai yaduwa, yana nufin cewa samfurin RNA na iya zama ƙasƙanci, kuma ana ba da shawarar yin amfani da hanyar da aka bayyana daga baya don ƙara gwada ingancin RNA.

Hoto 2. Kwatanta ƙasƙanci (layin 2) da RNA mara kyau (layin 3) akan agarose gel electrophoresis

03 Agilent Bioanalyzer

Baya ga hanyar agarose gel electrophoresis da aka bayyana a sama, wanda zai iya taimaka mana gano mutuncin RNA cikin sauƙi da sauri, muna kuma iya amfani da Agilent bioanalyzer don tantance amincin RNA.Yana amfani da hadewar microfluidics, capillary electrophoresis, da fluorescence don tantance taro da amincin RNA.Ta amfani da ginanniyar algorithm don nazarin bayanin martabar samfurin RNA, Agilent bioanalyzer na iya ƙididdige ƙimar ƙimar ƙimar RNA, Lamba Integrity RNA (nan gaba ana kiranta da RIN) [1].Girman darajar RIN, mafi girman amincin RNA (1 ya lalace sosai, 10 shine mafi cika).Wasu gwaje-gwajen da suka haɗa da RNA suna ba da shawarar amfani da RIN azaman ma'auni don ƙima mai inganci.Ɗaukar gwaje-gwaje masu girma-fisher (wanda ake kira NGS) a matsayin misali, jagororin Oncomine ™ Human Immune Repertoire, wanda ake amfani da shi don gano kwayoyin B da T cell antigen receptors a cikin Thermo Fisher's Oncomine panel, ya ba da shawarar cewa samfurori tare da ƙimar RIN mafi girma fiye da 4, Ƙarin ingantaccen karatu da clones za a iya auna 3).Akwai nau'ikan shawarwari daban-daban don bangarori daban-daban, kuma galibi mafi girma RIN na iya kawo bayanai masu inganci.

Hoto 3, a cikin gwaje-gwajen Repertoire na Oncomine™ Immune Repertoire, samfurori tare da RIN fiye da 4 na iya gano ƙarin ingantaccen karatu da clones T cell.【2】

Koyaya, ƙimar RIN shima yana da wasu iyakoki.Kodayake RIN yana da babban alaƙa tare da ingancin bayanan gwaji na NGS, bai dace da samfuran FFPE ba.An dade ana kula da samfuran FFPE ta hanyar sinadarai, kuma RNA da aka fitar gabaɗaya yana da ƙarancin ƙimar RIN.Koyaya, wannan ba yana nufin cewa ingantaccen bayanan gwajin dole ne ya zama mara gamsarwa ba.Don tantance ingancin samfuran FFPE daidai, muna buƙatar amfani da ma'auni ban da RIN.Baya ga RIN, Agilent bioanalyzer kuma yana iya ƙididdige ƙimar DV200 azaman ma'aunin ƙimar ingancin RNA.DV200 siga ce da ke ƙididdige rabon gutsuttsura sama da 200 bp a cikin samfurin RNA.DV200 shine mafi kyawun nuni na ingancin samfurin FFPE fiye da RIN.Ga RNA da FFPE ta fitar, tana da alaƙa mai zurfi da adadin ƙwayoyin halittar da za a iya gano su yadda ya kamata da kuma bambancin kwayoyin halitta [3].Ko da yake DV200 na iya daidaita ƙarancin gano ingancin FFPE, Agilent bioanalyzer har yanzu ba zai iya yin cikakken nazarin matsalolin ingancin samfuran RNA ba, gami da ko akwai masu hanawa a cikin samfuran.Masu hana kansu na iya yin tasiri ga haɓakar haɓakar gwaje-gwajen ƙasa kuma su rage adadin bayanai masu amfani.Don sanin ko akwai mai hanawa a cikin samfurin, zamu iya ɗaukar hanyar PCR mai kyalli na ainihin lokacin da aka kwatanta na gaba.

04 PCR mai kyalli na ainihin lokaci

Hanyar PCR mai kyalli na ainihi ba zai iya gano masu hanawa kawai a cikin samfurin ba, amma kuma yana nuna daidai ingancin RNA a cikin samfurin FFPE.Idan aka kwatanta da masu nazarin halittu na Agilent, ainihin kayan aikin ƙididdigewa na fluorescence sun fi shahara a manyan dakunan gwaje-gwajen halittu saboda faɗuwar aikace-aikacensu.Don gwada ingancin samfuran RNA, kawai muna buƙatar siyan ko shirya abubuwan bincike na farko don kwayoyin halitta na ciki, kamar GUSB (Cat no. Hs00939627).Ta yin amfani da wannan saitin firamare, bincike da ma'auni (jimlar RNA na sanannun maida hankali) don gudanar da cikakken gwaje-gwaje na ƙididdigewa, za a iya ƙididdige ingantaccen taro na RNA azaman ƙimar ƙimar ingancin RNA (Functional RNA Quantitation (FRQ) a takaice).A cikin gwajin NGS, mun gano cewa FRQ na samfuran RNA yana da alaƙa mai girma tare da ingantaccen ƙarar bayanai.Ga duk samfuran da suka fi 0.2ng/ul FRQ, aƙalla 70% na karatun na iya rufe tsarin tunani yadda ya kamata (Hoto 4).

Hoto 4, ƙimar FRQ da aka gano ta hanyar ƙididdigewa mai haske yana da alaƙa mai girma (R2>0.9) tare da ingantaccen bayanan da aka samu a cikin gwajin NGS.Layin ja shine ƙimar FRQ daidai da 0.2 ng/ul (log10 = -0.7).【4】

Baya ga kasancewa da amfani ga samfuran FFPE, ainihin lokacin ƙididdige hanyar PCR kuma na iya sa ido sosai ga masu hanawa a cikin samfuran.Zamu iya gano samfurin da za a gano cikin tsarin amsawa tare da ingantaccen iko na ciki (IPC) da assay, sannan kuma kuyi sulhu mai haske don samun darajar CT.Idan darajar Ct ta kasance a bayan ƙimar Ct a cikin abin da ba samfurin ba, yana nuna cewa mai hanawa yana cikin samfurin kuma ya hana haɓakar haɓakawa a cikin amsawa.

05 Hanyar launi mai kyalli na Qubit

Qubit Fluorometer ita ce ƙaramar na'urar da aka fi amfani da ita don tattarawar acid nucleic da gano tsafta, wanda ke da sauƙin aiki kuma yana wanzuwa a kusan kowane ɗakin binciken ƙwayoyin halitta.Yana ƙididdige yawan adadin nucleic acid daidai ta hanyar ganowa da rini mai ɗaure mai kyalli (Qubit detection reagent).Qubit yana da babban hankali da ƙayyadaddun ƙayyadaddun bayanai, kuma yana iya ƙididdige adadin RNA daidai zuwa matakin pg/µL.Baya ga sanannen ikon ƙididdige yawan adadin acid nucleic, sabon samfurin Thermo Fisher, Qubit 4.0, yana iya gano amincin RNA.Tsarin gano RNA na Qubit 4.0 (RNA IQ Assay) yana gano amincin RNA ta hanyar gano takamaiman rinayen kyalli guda biyu a lokaci guda.Wadannan rini mai kyalli guda biyu na iya ɗaure ga manyan gutsuttsura da ƙananan guntuwar RNA, bi da bi.Wadannan rini mai kyalli guda biyu suna nuna adadin manyan gutsuttsuran RNA a cikin samfurin, kuma daga wannan ana iya ƙididdige ƙimar IQ (Mutunci da Inganci) mai wakiltar ingancin RNA.Ƙimar IQ tana amfani da samfuran FFPE da waɗanda ba FFPE ba, kuma yana da tasiri mai girma akan ingancin jeri na gaba.Ɗaukar gwaje-gwajen NGS a matsayin misali, a cikin gwaje-gwajen gwajin RNA-Seq da aka yi akan dandamalin Ion torrent™, yawancin samfuran da ke da ƙimar IQ sama da 4 suna da aƙalla 50% ingantaccen karantawa (Hoto 5).Idan aka kwatanta da hanyoyin gano abubuwan da aka ambata a sama, Qubit IQ Assay ba wai kawai ya fi dacewa don aiki ba kuma yana ɗaukar ɗan lokaci kaɗan (a cikin mintuna biyar), amma kuma yana da babban alaƙa tsakanin ma'aunin ƙimar IQ da aka auna da ingancin bayanai na gwaje-gwaje na ƙasa.

Hoto na 5, akwai babban alaƙa tsakanin ƙimar Qubit RNA IQ da karanta taswira na RNA-Seq.【5】

Ta hanyar gabatarwar da ke sama, na yi imani cewa kowa yana da isasshen fahimtar hanyoyin sarrafa ingancin RNA daban-daban.A aikace, zaku iya zaɓarhanyar da ta dace bisa ga nau'in samfurin da kayan aikin da ake ciki.Ta hanyar sarrafa ingancin RNA da kyau za mu iya guje wa gazawar gwaje-gwajen da suka biyo baya sakamakon rashin ingancin samfurin, don haka adana lokaci, kuzari da tsada.

Kayayyakin Magana:

Jimlar Dabbobin Keɓewar RNA

Salon Warewa Kit ɗin RNA

nassoshi

【1】 Schroeder, A., Mueller, O., Stocker, S. et al.RIN: lambar mutuncin RNA don sanya ƙima ga ma'aunin RNA.BMC Molecular Biol 7, 3 (2006).https:// doi .org/10.1186/1471-21 99-7-3

【2】 Jagoran Mai Amfani da Maganganun Maganin Kariya na Mutum (Pub. No. MAN0017438 Rev. C.0).

Leah C Wehmas, Charles E Wood, Brian N Chorley, Carole L Yauk, Gail M Nelson, Susan D Hester. 73,https://doi.org/10.1093/toxsci/