Kuyaziwa ukuba kwimfundiso ephakathi, i-RNA ngumlamli obhaliweyo phakathi kwe-DNA kunye nokubonakaliswa kweprotheni.Xa kuthelekiswa nokufunyaniswa kwe-DNA, ukufunyaniswa kwe-RNA kunokubonakalisa ngokuthe ngqo ukubonakaliswa kofuzo kwizinto eziphilayo.Iimvavanyo ezibandakanya i-RNA zibandakanya: i-qRT-PCR, i-RNA-Seq, kunye nokufunyanwa kofuzo lwe-fusion, njl. Ngokusekelwe kwiimpawu ze-RNA ngokwayo (i-sugar ring ye-RNA ineqela elinye lamahhala le-hydroxyl kune-sugar ring ye-DNA), idityaniswe nenani elikhulu le-RNases kwimekobume, i-RNA ayizinzile kwaye kulula ukuthotywa kune-DNA.Udoti ngaphakathi, udoti ngaphandle, ukuba umgangatho we-RNA awulungile, ngoko iziphumo zovavanyo kufuneka zinganeliseki, zibonakaliswe ngokukodwa njengedatha engachanekanga okanye ukuphindaphinda kakubi.Ke ngoko, ingqwalasela engaphezulu kufuneka ihlawulwe ekusetyenzweni kwe-RNA, kwaye ikhonkco lolawulo lomgangatho likwabaluleke ngakumbi ukuqinisekisa ukuchaneka kunye nokuchaneka kwedatha yovavanyo elandelayo.

Kulawulo lomgangatho we-RNA, kukho iindlela eziqhelekileyo ezisetyenziswayo zilandelayo:

• I-Spectrophotometry
• i-agarose gel electrophoresis
• I-Agilent Bioanalyzer
• ixesha lokwenyani lefluorescent quantitative PCR
• Indlela yedayi ye-Qubit fluorescent

01 I-Spectrophotometry

I-RNA iye yadibanisa iibhondi eziphindwe kabini kwaye inencopho yokufunxa kumgama ongamaza angama-260nm.Ngokomthetho weLambert-Beer, sinokubala i-RNA yoxinaniso ukusuka kwincopho yokufunxa kwi-260nm.Ukongeza, sinokubala ukucoceka kwe-RNA ngokomlinganiselo we-260nm, 280nm kunye ne-230nm incopho yokufunxa.I-280nm kunye ne-230nm ziincopho zokufunxa iiprotheni kunye neemolekyuli ezincinci, ngokulandelanayo.Umlinganiselo we-A260 / A280 kunye ne-A260 / A230 yococeko olufanelekileyo lwe-RNA kufuneka ibe mkhulu kune-2. Ukuba ingaphantsi kwe-2, ithetha ukuba kukho iprotheni okanye i-molecule encinci yokungcola kwisampuli ye-RNA kwaye kufuneka ihlanjululwe kwakhona.Imithombo yongcoliseko iya kuchaphazela imifuniselo esezantsi, efana nokuthintela ulwazelelo lokwandiswa kweempendulo zePCR, okukhokelela kwiziphumo ezingachanekanga zobungakanani.Ukucoceka kwe-RNA kunempembelelo enkulu kwiziphumo ezilandelayo, ngoko ke i-spectrophotometry ngokuqhelekileyo ilikhonkco lokulawula umgangatho oyimfuneko kwinyathelo lokuqala kwiimvavanyo ze-nucleic acid.

Umzobo 1. I-RNA / DNA Absorption Spectrum eqhelekileyo

02 i-Agarose gel electrophoresis

Ukongeza kubunyulu, imfezeko ye-RNA sesinye sezalathisi ezibalulekileyo zokugweba umgangatho we-RNA.Ukuthotywa kwe-RNA kuya kukhokelela kwinani elikhulu lamaqhekeza amafutshane kwisampuli, ngoko ke inani leengqungquthela ze-RNA ezinokufunyanwa ngokufanelekileyo kwaye zigutyungelwe ngokulandelelana kwereferensi ziya kuncitshiswa.Ukunyaniseka kwe-RNA kunokujongwa nge-electrophoresis ye-RNA iyonke kwi-1% yejeli ye-agarose.Le ndlela inokumisela ijeli ngokwakho, okanye usebenzise i-E-Gel™ System esele yenziwe yovavanyo lokuthembeka.Ngaphezulu kwe-80% ye-RNA iyonke yi-ribosomal RNA, uninzi lwayo luqulunqwe yi-28S kunye ne-18S rRNA (kwiinkqubo zamamalian).Umgangatho olungileyo we-RNA uya kubonisa imivalo emibini ecacileyo eqaqambileyo, eyi-28S kunye ne-18S imivalo eqaqambileyo, ngokulandelelana, kwi-5 Kb kunye ne-2 Kb, kwaye umlinganiselo uya kuthanda ukuba usondele kwi-2: 1.Ukuba ikwimeko yokusabalalisa, kuthetha ukuba isampuli ye-RNA inokuba ithotyiwe, kwaye kuyacetyiswa ukuba kusetyenziswe indlela echazwe kamva ukuvavanya ngakumbi umgangatho we-RNA.

Umzobo 2. Ukuthelekiswa kokuthotywa (umzila we-2) kunye ne-RNA engaguqukiyo (i-layini ye-3) kwi-agarose gel electrophoresis

03 Agilent Bioanalyzer

Ukongeza kwindlela ye-agarose gel electrophoresis echazwe ngasentla, enokusinceda sibone ingqibelelo ye-RNA ngokulula kwaye ngokukhawuleza, sinokusebenzisa i-Agilent bioanalyzer ukumisela ingqibelelo ye-RNA.Isebenzisa indibaniselwano ye-microfluidics, i-capillary electrophoresis, kunye ne-fluorescence ukuvavanya ukugxila kwe-RNA kunye nokuthembeka.Ngokusebenzisa i-algorithm eyakhelwe-ngaphakathi ukuhlalutya iprofayili yesampuli ye-RNA, i-Agilent bioanalyzer ingabala i-reference ye-RNA yengqibelelo yexabiso, i-RNA Integrity Number (emva koku kuthiwa yi-RIN) [1].Ixabiso elikhulu le-RIN, iphezulu ingqibelelo ye-RNA (i-1 ithotywe ngokugqithiseleyo, i-10 iyona epheleleyo).Ezinye iimvavanyo ezibandakanya i-RNA zicebisa ukusebenzisa i-RIN njengepharamitha yovavanyo lomgangatho.Ukuthatha iimvavanyo zokulandelelana kwe-high-throughput (emva koku kubhekiselwa kuyo njenge-NGS) njengomzekelo, izikhokelo ze-Oncomine™ Human Immune Repertoire, esetyenziselwa ukufumanisa i-B cell kunye ne-T cell receptors ye-antigen kwi-Thermo Fisher's Oncomine panel series, iphakamisa ukuba iisampulu ezinexabiso le-RIN elikhulu kune-4, Ukufundwa okusebenzayo ngakumbi kunye ne-clones ingaba yi-3.Kukho uluhlu olwahlukeneyo olucetyiswayo lweepaneli ezahlukeneyo, kwaye kaninzi i-RIN ephezulu inokuzisa idatha esebenzayo ngakumbi.

Umzobo 3, kwiimvavanyo ze-Oncomine ™ Human Immune Repertoire, iisampuli ezine-RIN ezinkulu kune-4 zinokubona ukufundwa okusebenzayo ngakumbi kunye nee-T cell clones.【2】

Nangona kunjalo, ixabiso le-RIN linemida ethile.Nangona i-RIN inonxulumano oluphezulu nomgangatho wedatha yovavanyo ye-NGS, ayifanelekanga iisampulu ze-FFPE.Iisampulu ze-FFPE ziphathwe ngamachiza ixesha elide, kwaye i-RNA ekhutshiweyo ngokubanzi inexabiso eliphantsi le-RIN.Nangona kunjalo, oku akuthethi ukuba idatha esebenzayo yovavanyo kufuneka inganeliseki.Ukuvavanya ngokuchanekileyo umgangatho weesampuli zeFFPE, kufuneka sisebenzise imilinganiselo ngaphandle kwe-RIN.Ukongeza kwi-RIN, i-Agilent bioanalyzer inokubala ixabiso le-DV200 njengeparamitha yokuvavanya umgangatho we-RNA.I-DV200 yiparameter ebala umyinge wamaqhekeza amakhulu kuno-200 bp kwisampulu ye-RNA.I-DV200 luphawu olungcono lwesampulu ye-FFPE kune-RIN.Kwi-RNA ekhutshwe yi-FFPE, inonxulumano oluphezulu kakhulu kunye nenani lemizila yemfuza enokubonwa ngokufanelekileyo kunye nokwahlukana kwemfuza [3].Nangona i-DV200 inokwenza ukusilela ekufumaneni umgangatho we-FFPE, i-Agilent bioanalyzer isengakwazi ukuhlalutya ngokubanzi iingxaki zomgangatho kwiisampuli ze-RNA, kubandakanywa nokuba kukho inhibitors kwiisampuli.Ii-inhibitors ngokwazo zinokuchaphazela ukusebenza kakuhle kokukhulisa imifuniselo esezantsi kunye nokunciphisa inani ledatha eluncedo.Ukwazi ukuba ngaba kukho inhibitor kwisampulu, singasebenzisa indlela ye-PCR yexesha lokwenyani yobungakanani befluorescent echazwe ngokulandelayo.

04 ixesha lokwenyani lefluorescent quantitative PCR

Indlela ye-PCR yexesha lokwenyani ye-fluorescent ayinakukwazi ukubona kuphela ii-inhibitors kwisampulu, kodwa iphinda ibonise ngokuchanekileyo umgangatho we-RNA kwisampulu ye-FFPE.Xa kuthelekiswa nabahlalutyi bebhayoloji be-Agilent, izixhobo zobungakanani bexesha le-fluorescence zithandwa kakhulu kwiilabhoratri eziphambili zebhayoloji ngenxa yosetyenziso lwazo olubanzi.Ukuvavanya umgangatho weesampuli ze-RNA, kufuneka sithenge kuphela okanye silungiselele i-primer probes kwi-genesis yereferensi yangaphakathi, njenge-GUSB (Cat no. Hs00939627).Ngokusebenzisa le seti yee-primers, i-probes kunye nemigangatho (i-RNA iyonke yoxinaniso olwaziwayo) ukwenza iimvavanyo zobungakanani obupheleleyo, i-RNA fragment concentration esebenzayo ingabalwa njengomgangatho wokuvavanya umgangatho we-RNA (i-Functional RNA Quantitation (FRQ) ngokufutshane).Kuvavanyo lwe-NGS, sifumene ukuba i-FRQ yeesampulu ze-RNA inonxibelelwano oluphezulu kakhulu kunye nomthamo wedatha osebenzayo.Kuzo zonke iisampuli ezinkulu kune-0.2ng / uL FRQ, ubuncinane i-70% yokufundwa inokugubungela ngokufanelekileyo ukulandelelana kwereferensi (Umfanekiso 4).

Umzobo we-4, ixabiso le-FRQ elifunyenwe yi-fluorescence quantitative method ine-correlation ephezulu kakhulu (R2>0.9) kunye neenkcukacha ezisebenzayo ezifunyenwe kuvavanyo lwe-NGS.Umgca obomvu lixabiso le-FRQ elilingana ne-0.2 ng / uL (log10 = -0.7).【4】

Ukongeza ekusebenzeni kwiisampulu ze-FFPE, indlela yobungakanani bexesha lokwenyani ye-PCR inokubeka esweni ngokusebenzayo ii-inhibitors kwiisampulu.Singongeza isampuli ukuba ibhaqwe kwinkqubo reaction kunye Ulawulo Positive Ngaphakathi (IPC) kunye Assay yayo, kwaye ke wenze fluorescence quantification ukufumana ixabiso Ct.Ukuba ixabiso le-Ct lihlala emva kwexabiso le-Ct kwi-reaction ye-no-sample, libonisa ukuba i-inhibitor ikhona kwisampuli kwaye inqanda ukunyusa ukunyusa kwi-reaction.

05 Indlela yedayi ye-Qubit fluorescent

IQubit Fluorometer sesona sixhobo sincinci sisetyenziswa kakhulu soxinaniso lwe-nucleic acid kunye nokubhaqa ubunyulu, ekulula ukusebenza kwaye sikhona phantse kuyo yonke ilabhoratri yebhayoloji yemolekyuli.Ibala ngokuchanekileyo i-concentration ye-nucleic acid ngokufumanisa kunye ne-nucleic acid-binding fluorescent dye (Qubit discovery reagent).I-Qubit inovakalelo oluphezulu kunye neenkcukacha ezithile, kwaye inokulinganisa ngokuchanekileyo i-RNA ukuya kwi-pg/µL yoxinaniso.Ukongeza kwisakhono esaziwayo sokulinganisa ngokuchanekileyo i-nucleic acid concentration, imodeli entsha ye-Thermo Fisher entsha, i-Qubit 4.0, inokubona ukuthembeka kwe-RNA.I-Qubit 4.0's inkqubo yokufumanisa i-RNA (i-RNA IQ Assay) ibona ingqibelelo ye-RNA ngokubona ngaxeshanye iidayi ezimbini ezikhethekileyo ze-fluorescent.Ezi zimbini zedayi ze-fluorescent zinokubophelela kumaqhekeza amakhulu kunye namaqhekeza amancinci e-RNA, ngokulandelanayo.Ezi zimbini zedayi ze-fluorescent zibonisa umlinganiselo wamacandelo amakhulu e-RNA kwisampulu, kwaye ukusuka kule xabiso le-IQ (Integrity and Quality) emele umgangatho we-RNA ingabalwa.Ixabiso le-IQ lisebenza kuzo zombini iisampuli zeFFPE kunye ne-non-FFPE, kwaye inempembelelo enkulu kumgangatho wokulandelelana okulandelayo.Ukuthatha imifuniselo ye-NGS njengomzekelo, kwiimvavanyo zovavanyo ze-RNA-Seq ezenziwe kwiqonga le-Ion torrent™, iisampulu ezininzi ezinamaxabiso e-IQ amakhulu kune-4 zineepesenti ezingama-50 zokufundwa okusebenzayo (Umfanekiso 5).Xa kuthelekiswa neendlela zokufumanisa ezikhankanywe ngasentla, i-Qubit IQ Assay ayifanelekanga nje ukuba isebenze kwaye ithatha ixesha elincinci (ngaphakathi kwemizuzu emihlanu), kodwa inokulungelelaniswa okukhulu phakathi kwexabiso le-parameter elinganisiweyo ye-IQ kunye nomgangatho wedatha yezilingo ezisezantsi.

Umzobo we-5, kukho ulungelelwaniso olukhulu phakathi kwexabiso le-Qubit RNA IQ kunye nemephu yokufunda ye-RNA-Seq.【5】

Ngentshayelelo engentla, ndikholelwa ukuba wonke umntu unokuqonda okwaneleyo kweendlela ezahlukeneyo zokulawula umgangatho we-RNA.Ngokwenza, unokukhethaindlela ehambelanayo ngokohlobo lwesampulu kunye nezixhobo ezikhoyo.Kuphela ngokulawula umgangatho we-RNA kakuhle sinokukuphepha ukusilela kovavanyo olulandelayo olubangelwa ngumgangatho wesampulu engafanelekanga, ngaloo ndlela songa ixesha elixabisekileyo, amandla kunye neendleko.

Iimveliso zeReferensi:

Animal Total RNA Isolation Kit

ISeli iyonke yeRNA Isolation Kit

iimbekiselo

【1】Schroeder, A., Mueller, O., Stocker, S. et al.I-RIN: inombolo yengqibelelo ye-RNA yokwabela amaxabiso emfezeko kwimilinganiselo ye-RNA.I-BMC ye-Molecular Biol 7, 3 (2006).NONE// doi .org/10.1186/1471-21 99-7-3

【2】Isikhokelo somsebenzisi se-Oncomine Human Immune Repertoire (Pub. No. MAN0017438 Rev. C.0).

【3】 ULeah C Wehmas, uCharles E Wood, uBrian N Chorley, uCarole L Yauk, uGail M Nelson, uSusan D Hester, iiMetrikhi zoMgangatho owomeleziweyo wokuHlola i-RNA ephuma kwi-Archival Formalin-Fixed-Embedded Tissue Samples, i-Toxicological Sciences, Volume 3720, Agasti 3720, Agasti 3720https://doi.org/10.1093/toxsci/