A maara nke ọma na na n'etiti ozizi, RNA bụ transcriptional ogbugbo n'etiti DNA na protein okwu.E jiri ya tụnyere nchọpụta DNA, nchọpụta RNA nwere ike na-egosipụta nke ọma n'ụdị mkpụrụ ndụ ihe nketa dị n'ime ihe ndị dị ndụ.Nnwale metụtara RNA gụnyere: qRT-PCR, RNA-Seq, na nchọpụta mkpụrụ ndụ fusion, wdg Dabere na njirimara nke RNA n'onwe ya (mgbanaka shuga nke RNA nwere otu hydroxyl n'efu karịa mgbanaka shuga nke DNA), yana ọnụ ọgụgụ RNases dị ukwuu na gburugburu ebe obibi, RNA na-adịghị akwụsi ike ma dị mfe karịa DNA.Ntufu na, mkpofu, ma ọ bụrụ na ogo RNA adịghị mma, mgbe ahụ nsonaazụ nnwale ga-abụ nke na-adịghị mma, gosipụtara kpọmkwem dị ka data na-ezighi ezi ma ọ bụ enweghị ike ikwughachi.Ya mere, ekwesịrị itinye nlebara anya na nhazi nke RNA, yana njikọ nke njikwa mma dịkwa mkpa iji hụ na izi ezi na izi ezi nke data nnwale na-esote.

Maka njikwa mma nke RNA, a na-enwekarị usoro ndị a na-ejikarị:

• Spectrophotometry
• agarose gel electrophoresis
• Agilent Bioanalyzer
• ezigbo oge fluorescent quantitative PCR
• Usoro agba agba qubit

01 Spectrophotometry

RNA ejikọtala agbụ abụọ ma nwee ọnụ ọgụgụ kasị elu nke mmịnye n'ogologo 260nm.Dịka iwu Lambert-Beer siri dị, anyị nwere ike gbakọọ mkpokọta RNA site na ọnụ ọgụgụ kacha elu nke nnabata na 260nm.Na mgbakwunye, anyị nwekwara ike gbakọọ ịdị ọcha nke RNA dị ka oke nke 260nm, 280nm na 230nm absorption kacha.280nm na 230nm bụ ọnụ ọgụgụ kasị elu nke protein na obere ụmụ irighiri ihe, n'otu n'otu.Oke nke A260/A280 na A260/A230 nke ruru eru ịdị ọcha RNA kwesịrị ịdị ukwuu karịa 2. Ọ bụrụ na ọ na-erughị 2, ọ pụtara na enwere protein ma ọ bụ obere nje molecule na nlele RNA na ọ dị mkpa ka a sachaa ọzọ.Isi mmalite mmetọ ga-emetụta nnwale ndị dị n'okpuru ala, dị ka igbochi nrụpụta arụmọrụ nke mmeghachi omume PCR, na-ebute nsonaazụ ọnụọgụ ezighi ezi.Ịdị ọcha nke RNA nwere mmetụta dị ukwuu na nsonaazụ ndị na-esote, yabụ spectrophotometry bụ n'ozuzu njikọ njikwa mma dị mkpa na nzọụkwụ mbụ na nnwale nucleic acid.

Ọgụgụ 1. Ụdị RNA/DNA Absorption Spectrum

02 Agarose gel electrophoresis

Na mgbakwunye na ịdị ọcha, iguzosi ike n'ezi ihe nke RNA bụkwa otu n'ime ihe ndị dị mkpa maka ikpebi ogo RNA.Nbibi nke RNA ga-eduga na ọnụ ọgụgụ dị mkpirikpi nke mpempe akwụkwọ dị na nlele ahụ, ya mere ọnụ ọgụgụ nke RNA nke nwere ike ịchọta nke ọma ma kpuchie ya site na usoro ntụaka ga-ebelata.Enwere ike ịlele iguzosi ike n'ezi ihe RNA site na electrophoresis nke mkpokọta RNA na gel agarose 1%.Usoro a nwere ike hazie jel n'onwe gị, ma ọ bụ jiri Sistemụ E-Gel™ emebere nke ọma maka nnwale iguzosi ike n'ezi ihe.Ihe karịrị 80% nke mkpokọta RNA bụ ribosomal RNA, ọtụtụ n'ime ha bụ 28S na 18S rRNA (na sistemu anụ ahụ).RNA dị mma ga-egosi ogwe abụọ na-egbuke egbuke, nke bụ 28S na 18S na-egbuke egbuke, n'otu n'otu, na 5 Kb na 2 Kb, na nha ga-adị nso na 2: 1.Ọ bụrụ na ọ nọ n'ọnọdụ na-agbasa, ọ pụtara na ihe nlele RNA nwere ike mebie ya, a na-atụ aro ka iji usoro akọwara emechaa iji nwalee ogo RNA ọzọ.

Ọgụgụ 2. Ntụle nke degraded (ụzọ 2) na RNA na-emebibeghị (ụzọ 3) na agarose gel electrophoresis

03 Agilent Bioanalyzer

Na mgbakwunye na usoro agarose gel electrophoresis akọwara n'elu, nke nwere ike inyere anyị aka ịmata iguzosi ike n'ezi ihe nke RNA ngwa ngwa na ngwa ngwa, anyị nwekwara ike iji Agilent bioanalyzer iji chọpụta iguzosi ike n'ezi ihe nke RNA.Ọ na-eji ngwakọta nke microfluidics, capillary electrophoresis, na fluorescence iji nyochaa ntinye RNA na iguzosi ike n'ezi ihe.Site n'iji algọridim arụnyere arụnyere iji nyochaa profaịlụ nke ihe nlele RNA, Agilent bioanalyzer nwere ike gbakọọ uru iguzosi ike n'ezi ihe RNA, Nọmba Integrity RNA (nke a na-akpọ RIN) [1].Ka uru RIN buru ibu, na-abawanye iguzosi ike n'ezi ihe nke RNA (1 na-eweda ya nke ukwuu, 10 bụ nke zuru oke).Ụfọdụ nnwale metụtara RNA na-atụ aro iji RIN dị ka ihe nleba anya maka nleba anya.Inweta nnwale nke usoro ihe omume dị elu (nke a na-akpọ NGS) dị ka ọmụmaatụ, ntuziaka nke Oncomine ™ Human Immune Repertoire, nke a na-eji achọpụta ndị na-anabata sel B na T cell na Thermo Fisher's Oncomine panel, na-atụ aro na ihe atụ nwere ụkpụrụ RIN karịrị 4, enwere ike ịgụta nke ọma na mmechi.Enwere ọkwa dị iche iche akwadoro maka akụkụ dị iche iche, na mgbe mgbe RIN dị elu nwere ike iweta data dị irè karị.

Ọgụgụ 3, na nyocha Oncomine™ Human Immune Repertoire, ihe nlele nwere RIN karịrị 4 nwere ike ịchọpụta agụ dị mma yana clones T cell.【2】

Agbanyeghị, uru RIN nwekwara oke ụfọdụ.Ọ bụ ezie na RIN nwere njikọ dị elu na ogo data nnwale NGS, ọ dabaraghị maka nlele FFPE.A na-agwọta ihe nlele FFPE na kemịkalụ ogologo oge, na RNA ewepụtara n'ozuzu nwere uru RIN dị obere.Otú ọ dị, nke a apụtaghị na data dị irè nke nnwale ahụ aghaghị ịbụ ihe na-adịghị mma.Iji nyochaa n'ụzọ ziri ezi àgwà nke nlele FFPE, anyị kwesịrị iji nha na-abụghị RIN.Na mgbakwunye na RIN, Agilent bioanalyzer nwekwara ike gbakọọ uru DV200 dị ka ihe nleba anya nke ogo RNA.DV200 bụ oke nke na-agbakọ oke nke iberibe ihe karịrị 200 bp na nlele RNA.DV200 bụ ihe ngosi ka mma nke ụdị nlele FFPE karịa RIN.Maka RNA nke FFPE weputara, o nwere njikọ dị oke elu na ọnụọgụ mkpụrụ ndụ ihe nketa nwere ike ịchọpụta nke ọma yana ụdị mkpụrụ ndụ ihe dị iche iche [3].Ọ bụ ezie na DV200 nwere ike mejupụta erughị eru dị na nchọta ịdịmma nke FFPE, Agilent bioanalyzer enweghị ike nyochaa nke ọma nsogbu dị na nlele RNA, gụnyere ma enwere ihe mgbochi na nlele.Ndị na-egbochi onwe ha nwere ike imetụta mmụba arụmọrụ nke nyocha ala ma belata ọnụọgụ data bara uru.Iji mara ma enwere ihe mgbochi na ihe nlele ahụ, anyị nwere ike ịnwe ezigbo oge fluorescent quantitative usoro PCR akọwara na-esote.

04 ezigbo oge fluorescent quantitative PCR

Usoro PCR nke ezigbo fluorescent quantitative nwere ike ọ bụghị naanị ịchọpụta ihe mgbochi na nlele ahụ, kamakwa gosipụta n'ụzọ ziri ezi ogo RNA na nlele FFPE.E jiri ya tụnyere Agilent biological analyzers, ezigbo oge fluorescence quantitative ngwa na-ewu ewu na nnukwu ụlọ nyocha ihe ndụ n'ihi ngwa ha sara mbara.Iji nwalee ogo nke ihe nlele RNA, naanị anyị kwesịrị ịzụrụ ma ọ bụ dozie nyocha primer maka mkpụrụ ndụ ihe n'ime, dị ka GUSB (Cat no. Hs00939627).Site na iji setịpụ nke primers, nyocha na ụkpụrụ (ngụkọta RNA nke mkpokọta amaara ama) iji mee nnwale ọnụọgụ zuru oke, enwere ike gbakọọ mkpokọta RNA dị irè dị ka ọkọlọtọ nyocha nke ogo RNA (Functional RNA Quantitation (FRQ) maka mkpụmkpụ).N'ime ule NGS, anyị chọpụtara na FRQ nke nlele RNA nwere njikọ dị oke elu na oke data dị irè.Maka nlele niile karịrị 0.2ng/ul FRQ, opekata mpe 70% nke ndị a na-agụ nwere ike kpuchie usoro ntụnye aka nke ọma (Ọnụ ọgụgụ 4).

Ọnụọgụgụ 4, uru FRQ nke usoro fluorescence quantitative achọpụtara nwere njikọ dị oke elu (R2>0.9) yana data dị irè enwetara na nnwale NGS.Ahịrị uhie bụ uru FRQ ruru 0.2 ng/ul (log10 = -0.7).【4】

Na mgbakwunye na ọdabara na nlele FFPE, usoro PCR nke na-eme n'ezie nwekwara ike nyochaa ndị na-egbochi ihe n'omume.Anyị nwere ike ịgbakwunye ihe nlele a ga-achọpụta n'ime usoro mmeghachi omume na njikwa dị mma nke ime (IPC) na nyocha ya, wee mee ọnụọgụ fluorescence iji nweta uru Ct.Ọ bụrụ na uru Ct dị n'azụ uru Ct na mmeghachi omume na-adịghị, ọ na-egosi na onye na-emechi ihe dị na ihe nlele ahụ ma na-egbochi mmepụta nkwalite na mmeghachi omume.

05 Qubit fluorescent usoro agba agba

Qubit Fluorometer bụ obere ngwaọrụ a na-ejikarị eme ihe maka ntinye uche nke acid nucleic na ịchọpụta ịdị ọcha, nke dị mfe iji rụọ ọrụ ma dị n'ihe fọrọ nke nta ka ọ bụrụ ụlọ nyocha ihe ọmụmụ mkpụrụ ndụ ọ bụla.Ọ na-agbakọ n'ụzọ ziri ezi ntinye nke nucleic acid site na ịchọpụta na nucleic acid na-ejikọta fluorescent dye (Qubit detection reagent).Qubit nwere mmetụta dị elu na nkọwapụta, ma nwee ike ịkọwapụta RNA n'ụzọ ziri ezi ruo na ntinye pg/µL.Na mgbakwunye na ikike amara nke ọma ịkọwapụta mkpokọta acid nucleic nke ọma, ụdị ọhụrụ Thermo Fisher, Qubit 4.0, nwekwara ike ịchọpụta iguzosi ike n'ezi ihe nke RNA.Sistemụ nchọpụta RNA nke Qubit 4.0 (RNA IQ Assay) na-achọpụta iguzosi ike n'ezi ihe nke RNA site n'ịchọpụta ihe eji esiji fluorescent abụọ n'otu oge.Ejiji fluorescent abụọ a nwere ike jikọta na nnukwu iberibe na obere iberibe RNA, n'otu n'otu.Achaji fluorescent abụọ a na-egosi oke nke nnukwu iberibe RNA dị na nlele ahụ, site na nke a, enwere ike gbakọọ uru IQ (Ikwesị ntụkwasị obi na Ogo) nke na-anọchite anya ogo RNA.Uru IQ na-adabara ma ụdị FFPE na ndị na-abụghị FFPE, ma nwee mmetụta dị ukwuu na ogo usoro na-esote.Inweta nnwale NGS dị ka ọmụmaatụ, na nnwale nnwale RNA-Seq emere n'elu ikpo okwu Ion torrent ™, ọtụtụ ihe nlele nwere ụkpụrụ IQ karịrị 4 nwere opekata mpe 50% na-agụ nke ọma (Nyocha 5).E jiri ya tụnyere ụzọ nchọpụta ndị a kpọtụrụ aha n'elu, Qubit IQ Assay abụghị naanị na-adaba adaba iji rụọ ọrụ ma na-ewe obere oge (n'ime nkeji ise), kamakwa ọ nwere njikọ dị ukwuu n'etiti uru IQ tụrụ atụ na ogo data nke nnwale mgbada.

Onyonyo 5, enwere nnukwu njikọ dị n'etiti uru Qubit RNA IQ na eserese nke RNA-Seq.【5】

Site na mmeghe a dị n'elu, ekwenyere m na onye ọ bụla nwere nghọta zuru oke maka ụzọ njikwa ogo RNA dị iche iche.Na omume, ị nwere ike họrọusoro kwekọrọ dị ka ụdị ihe atụ na ngwá ọrụ dị ugbu a.Naanị site n'ịchịkwa àgwà RNA nke ọma, anyị nwere ike izere ọdịda nke nnwale ndị na-esote nke àgwà adịghị mma kpatara, si otú a na-echekwa oge, ike na ọnụ ahịa bara uru.

Ngwaahịa ntụaka:

Ngwakọta Mkpokọta RNA anụmanụ

Ngwakọta mkpokọta cell RNA iche

ntụaka

【1】 Schroeder, A., Mueller, O., Stocker, S. et al.RIN: nọmba iguzosi ike n'ezi ihe RNA maka ikenye ụkpụrụ iguzosi ike n'ezi ihe na nha RNA.BMC Molecular Biol 7, 3 (2006).https:// doi .org/10.1186/1471-21 99-7-3

【2】Ntụziaka onye ọrụ Oncomine Human Immune Repertoire (Pub. No. MAN0017438 Rev. C.0).

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