Birazwi neza ko muri dogma rwagati, RNA ni umuhuza wandikirana hagati ya ADN n'imvugo ya poroteyine.Ugereranije no kumenya ADN, gutahura RNA birashobora kwerekana neza imiterere ya gene mubinyabuzima.Ubushakashatsi burimo RNA burimo: qRT-PCR, RNA-Seq, hamwe no kumenya gene ya fusion, nibindi. Ukurikije ibiranga RNA ubwayo (impeta yisukari ya RNA ifite itsinda rimwe rya hydroxyl ryisanzuye kuruta isukari ya ADN), ifatanije numubare munini wa RNase mubidukikije, RNA irahungabana kandi byoroshye kwangirika kuruta ADN.Imyanda muri, imyanda hanze, niba ubwiza bwa RNA butameze neza, noneho ibisubizo byubushakashatsi bigomba kuba bidashimishije, bigaragazwa nkamakuru atariyo cyangwa gusubiramo nabi.Kubwibyo, hakwiye kwitabwaho cyane mugutunganya RNA, kandi ihuriro ryo kugenzura ubuziranenge naryo ni ngombwa kugirango hamenyekane neza amakuru yukuri yubushakashatsi.

Kugenzura ubuziranenge bwa RNA, muri rusange hari uburyo bukurikira bukoreshwa:

• Spectrophotometry
• agarose gel electrophorei
• Agilent Bioanalyser
• igihe nyacyo fluorescent yuzuye PCR
• Uburyo bwo gusiga irangi rya fluorescent

01 Spectrophotometry

RNA yahujije imigozi ibiri kandi ifite impinga yo kwinjirira ku burebure bwa 260nm.Dukurikije amategeko ya Lambert-Byeri, turashobora kubara ubunini bwa RNA duhereye ku gipimo cyo kwinjiza kuri 260nm.Mubyongeyeho, dushobora kandi kubara ubuziranenge bwa RNA dukurikije igipimo cya 260nm, 280nm na 230nm yo kwinjirira.280nm na 230nm ni impinga yo kwinjiza poroteyine na molekile nto.Ikigereranyo cya A260 / A280 na A260 / A230 cyujuje ubuziranenge bwa RNA kigomba kuba kirenze 2. Niba kiri munsi ya 2, bivuze ko hari proteine ​​cyangwa molekile ntoya yanduye murugero rwa RNA kandi igomba kongera kwezwa.Inkomoko yanduye izagira ingaruka kubigeragezo byo hasi, nko kubuza amplifisione ya reaction ya PCR, bikavamo ibisubizo bitari byo.Isuku ya RNA igira uruhare runini kubisubizo byakurikiyeho, bityo spekitifotometometrie muri rusange ni ihuriro ryingirakamaro mu kugenzura ubuziranenge mu ntambwe yambere mu bushakashatsi bwa acide nucleique.

Igicapo 1. Ibisanzwe RNA / ADN Absorption Spectrum

02 Agarose gel electrophorei

Usibye ubuziranenge, ubunyangamugayo bwa RNA nabwo ni kimwe mu bipimo by'ingenzi byo gusuzuma ubuziranenge bwa RNA.Kwangirika kwa RNA bizaganisha ku mubare munini wibice bigufi murugero, bityo umubare wibice bya RNA ushobora gutahurwa neza kandi ugakurikiranwa nurutonde rwerekanwe bizagabanuka.Ubusugire bwa RNA bushobora kugenzurwa na electrophorei ya RNA yose kuri gel 1% ya agarose.Ubu buryo bushobora gushiraho gel wenyine, cyangwa ugakoresha E-Gel ™ Sisitemu yo kugerageza ubunyangamugayo.Kurenga 80% ya RNA yose ni ribosomal RNA, inyinshi muri zo zigizwe na 28S na 18S rRNA (muri sisitemu y’inyamabere).Ubwiza bwiza RNA izerekana utubari tubiri tugaragara, aribwo 28S na 18S ibibari byaka, kuri 5 Kb na 2 Kb, kandi igipimo kizaba hafi ya 2: 1.Niba ari muburyo bukwirakwira, bivuze ko icyitegererezo cya RNA gishobora kuba cyarangiritse, kandi birasabwa gukoresha uburyo bwasobanuwe nyuma kugirango tumenye neza ubwiza bwa RNA.

Igishushanyo 2. Kugereranya ibyangiritse (umurongo wa 2) na RNA idahwitse (umurongo wa 3) kuri agarose gel electrophorei

03 Agilent Bioanalyser

Usibye uburyo bwa agarose gel electrophoreis yasobanuwe haruguru, ishobora kudufasha kumenya ubusugire bwa RNA mu buryo bwihuse kandi bwihuse, dushobora kandi gukoresha Agilent bioanalyser kugirango tumenye ubusugire bwa RNA.Ikoresha uruvange rwa microfluidics, capillary electrophoresis, na fluorescence kugirango isuzume ubunini bwa RNA nubunyangamugayo.Ukoresheje algorithm yubatswe kugirango isesengure imiterere yicyitegererezo cya RNA, Agilent bioanalyser irashobora kubara agaciro ka RNA ubunyangamugayo, Umubare wuburinganire bwa RNA (aha ni ukuvuga RIN) [1].Ninini agaciro ka RIN, niko ubunyangamugayo bwa RNA (1 bwangiritse cyane, 10 nabwo bwuzuye).Ubushakashatsi bumwe burimo RNA bwerekana gukoresha RIN nkibipimo byo gusuzuma ubuziranenge.Dufashe urugero rwinshi rwakurikiranye (nyuma yiswe NGS) nkurugero, umurongo ngenderwaho wa Oncomine ™ Human Immune Repertoire, ikoreshwa mugutahura reseptor ya B selile na T selile ya antigen yakira muri Thermo Fisher's Oncomine panel, byerekana ko ingero zifite agaciro ka RIN zirenze 4, Gusoma neza na clone bishobora gupimwa (Ishusho 3).Hano haribisabwa bitandukanye kubice bitandukanye, kandi akenshi RIN yo hejuru irashobora kuzana amakuru meza.

Igishushanyo cya 3, muri Oncomine ™ Ubushakashatsi bwabantu Immune Repertoire, ingero hamwe na RIN zirenze 4 zirashobora kumenya neza gusoma neza na T selile.【2】

Ariko, agaciro RIN nayo ifite aho igarukira.Nubwo RIN ifitanye isano ryinshi nubwiza bwamakuru yubushakashatsi bwa NGS, ntibikwiye kuburugero rwa FFPE.Ingero za FFPE zimaze igihe kinini zivurwa, kandi RNA yakuwe muri rusange ifite agaciro gake RIN.Ariko, ibi ntibisobanura ko amakuru yingirakamaro yubushakashatsi agomba kuba adashimishije.Kugirango dusuzume neza ubuziranenge bwa FFPE, dukeneye gukoresha ibipimo bitari RIN.Usibye RIN, Agilent bioanalyser irashobora kandi kubara agaciro ka DV200 nkibipimo byo gusuzuma ubuziranenge bwa RNA.DV200 ni ibipimo bibara igipimo cyibice birenga 200 bp murugero rwa RNA.DV200 nikimenyetso cyiza cyerekana icyitegererezo cya FFPE kuruta RIN.Kuri RNA yakuweho na FFPE, ifitanye isano rya bugufi n'umubare w'ingirabuzima fatizo zishobora kumenyekana neza ndetse n'ubwoko butandukanye bwa gen [3].Nubwo DV200 ishobora kuzuza ibitagenda neza mugutahura ubuziranenge bwa FFPE, bioanalyser ya Agilent ntishobora gusesengura byimazeyo ibibazo byubuziranenge mubitegererezo bya RNA, harimo no kumenya niba muri izo ngero harimo inzitizi.Inhibitor ubwayo irashobora kugira ingaruka kumikorere ya amplification yubushakashatsi bwo hasi kandi ikagabanya umubare wamakuru yingirakamaro.Kugirango tumenye niba hari inhibitor murugero, turashobora gukoresha igihe nyacyo fluorescent numero PCR yasobanuwe ubutaha.

04 igihe nyacyo fluorescent yuzuye PCR

Uburyo nyabwo bwa fluorescent numero ya PCR ntishobora kumenya gusa inhibitori murugero, ariko kandi irerekana neza ubwiza bwa RNA murugero rwa FFPE.Ugereranije nisesengura ryibinyabuzima bya Agilent, ibikoresho nyabyo bya fluorescence mugihe gikunzwe cyane muri laboratoire nini yibinyabuzima bitewe nuburyo bukoreshwa cyane.Kugirango tumenye ubuziranenge bw'icyitegererezo cya RNA, dukeneye gusa kugura cyangwa gutegura primer probe ya gen yerekanwe imbere, nka GUSB (Injangwe no Hs00939627).Ukoresheje iyi progaramu ya primers, probe hamwe nubuziranenge (RNA yuzuye yibitekerezo bizwi) kugirango ukore igeragezwa ryuzuye, kwibumbira hamwe kwa RNA birashobora kubarwa nkurwego rwo gusuzuma ubuziranenge bwa RNA (Imikorere ya RNA Quantitation (FRQ) mugihe gito).Mu kizamini cya NGS, twasanze FRQ yintangarugero ya RNA ifitanye isano nini cyane nubunini bwamakuru.Kuburugero rwose rurenze 0.2ng / uL FRQ, byibuze 70% byabasomwe birashobora gukwirakwiza neza urutonde (Ishusho 4).

Igishushanyo 4, agaciro ka FRQ kagaragajwe nuburyo bwo kubara bwa fluorescence bufite isano iri hejuru cyane (R2> 0.9) hamwe namakuru meza yabonetse mubushakashatsi bwa NGS.Umurongo utukura nigiciro cya FRQ kingana na 0.2 ng / uL (log10 = -0.7).【4】

Usibye gukoreshwa mubitegererezo bya FFPE, uburyo nyabwo bwa PCR burigihe burashobora no kugenzura neza inhibitor muri sample.Turashobora kongeramo icyitegererezo kugirango kigaragare muri sisitemu yogukora hamwe na Internal Positive Control (IPC) hamwe na Assay yayo, hanyuma dukore igipimo cya fluorescence kugirango tubone agaciro ka Ct.Niba agaciro ka Ct gasigaye inyuma ya Ct agaciro muri no-sample reaction, byerekana ko inhibitor ihari murugero kandi ikabuza amplification imikorere muri reaction.

05 Uburyo bwo gusiga irangi rya fluorescent

Qubit Fluorometer nigikoresho gikunze gukoreshwa mugukoresha aside nucleic aside hamwe no kumenya neza, byoroshye gukora kandi bibaho muri laboratoire ya biologiya hafi ya yose.Iharura neza ubunini bwa acide nucleic mu kumenya no gusiga irangi rya nucleic aside ihuza irangi (Qubit detection reagent).Qubit ifite sensibilité nini kandi yihariye, kandi irashobora kugereranya neza RNA kugeza kuri pg / µL.Usibye ubushobozi buzwi bwo kugereranya neza aside nucleique, urugero rushya rwa Thermo Fisher, Qubit 4.0, rushobora no kumenya ubusugire bwa RNA.Qubit 4.0′s Sisitemu yo gutahura RNA (RNA IQ Assay) itahura ubusugire bwa RNA icyarimwe ikamenya amarangi abiri yihariye ya fluorescent.Aya marangi abiri ya fluorescent arashobora guhuza ibice binini nuduce duto twa RNA.Aya marangi abiri ya fluorescent yerekana igipimo cyibice binini bya RNA murugero, kandi uhereye kuri agaciro IQ (Ubunyangamugayo nubuziranenge) byerekana ubuziranenge bwa RNA irashobora kubarwa.Agaciro ka IQ karakoreshwa kuri byombi bya FFPE na non-FFPE, kandi bifite ingaruka zikomeye kumiterere ikurikiranye.Dufashe urugero rwa NGS nk'urugero, mu bushakashatsi bwa RNA-Seq bwakorewe ku rubuga rwa Ion torrent ™, ingero nyinshi zifite agaciro ka IQ zirenze 4 zari zifite byibuze gusoma 50% (Ishusho 5).Ugereranije nuburyo bwo gutahura bwavuzwe haruguru, Qubit IQ Assay ntabwo yorohewe gusa gukora kandi ifata igihe gito (muminota itanu), ariko kandi ifite isano ikomeye hagati yikigereranyo cyapimwe IQ agaciro nubuziranenge bwamakuru yubushakashatsi bwo hasi.

Igicapo 5, hariho ihuriro rikomeye hagati ya Qubit RNA IQ agaciro nigishushanyo cyasomwe cya RNA-Seq.【5】

Binyuze mu ntangiriro yavuzwe haruguru, nizera ko buriwese afite gusobanukirwa bihagije kuburyo butandukanye bwo kugenzura ubuziranenge bwa RNA.Mu myitozo, urashobora guhitamouburyo bukwiranye ukurikije ubwoko bw'icyitegererezo n'ibikoresho bihari.Gusa mugucunga ubuziranenge bwa RNA neza dushobora kwirinda kunanirwa nubushakashatsi bwakurikiyeho buterwa nubwiza bwikitegererezo, bityo tukabika umwanya, imbaraga nigiciro.

Ibicuruzwa byerekana:

Inyamaswa Igiteranyo Cyuzuye RNA

Akagari Igiteranyo cya RNA Igikoresho cyo kwigunga

Indanganturo

【1】 Schroeder, A., Mueller, O., Ububiko, S. n'abandi.RIN: inomero ya RNA yo kugenera indangagaciro zuzuye mubipimo bya RNA.BMC Molecular Biol 7, 3 (2006).https:// doi .org / 10.1186 / 1471-21 99-7-3

【2】 Oncomine Umuntu Immune Repertoire Yumukoresha (Igitabo No MAN0017438 Ibyah C.0)

.https://doi.org/10.1093/toxsci/