I-TIANSeq RNA Ubuhlalu obuhlanzekile

Njengamanje, ubuchwepheshe bokulandelana okuphezulu kakhulu bususelwa kubuchwepheshe bokulandelana kwesizukulwane esilandelayo. Njengoba ubude bokufunda bobuchwepheshe bokulandelana besizukulwane esilandelayo bunqunyelwe, kufanele sihlukanise ukulandelana kobude obugcwele kube yimitapo yolwazi emincane ilandelane. Ngokuya ngezidingo zokuhlolwa okulandelanayo okuhlukile, imvamisa sikhetha ukulandelana okuphela okukodwa noma ukulandelana okuphela kabili. Njengamanje izingcezu ze-DNA zelabhulali yokulandelana kwesizukulwane esilandelayo zivame ukusatshalaliswa ebangeni lama-200-800 bp.

a) I-DNA iphansi futhi iqukethe ama-inhibitors. Sebenzisa amasampula wekhwalithi ephezulu ye-DNA ukugwema ukuvinjelwa komsebenzi we-enzyme.

b) Inani lesampula le-DNA alanele uma usebenzisa indlela engenayo i-PCR ukwakha umtapo wolwazi we-DNA. Lapho okokufaka kwe-DNA ehlukanisiwe kudlula ama-50 ng, ukuhamba komsebenzi okungenayo i-PCR kungenziwa ngokukhetha ngesikhathi senqubo yokwakhiwa kwelabhulali. Uma inombolo yekhophi yelabhulali iphansi kakhulu ukuthi ingalandelwa ngqo, umtapo wolwazi we-DNA ungakhuliswa yi-PCR ngemuva kwe-adaptha ligation.

c) Ukungcoliswa kwe-RNA kuholela ekungcolisweni kokuqala okungalungile kwe-DNA quantification RNA kungenzeka kube khona ngenqubo yokuhlanzwa kwe-genomic DNA, engaholela ekubalweni okungalungile kwe-DNA nasekulayisheni okwanele kwe-DNA ngesikhathi kwakhiwa umtapo wolwazi. I-RNA ingasuswa ngokwelapha nge-RNase.

A-1

a) Izingcezu ezincane (60 bp-120 bp) ziyavela izingcezu ezincane zivame ukuba izingcezu ze-adaptha noma izinciphisi ezakhiwe ngama-adapters. Ukuhlanzwa nge-Agencourt AMPure XP ubuhlalu bukazibuthe kungasusa ngempumelelo lezi zingcezu ze-adaptha futhi kuqinisekise ikhwalithi yokulandelana.

b) Izingcezu ezinkulu ziyavela emtatsheni wezincwadi ngemuva kokukhuliswa kwe-PCR Usayizi wocezu lwelabhulali ye-DNA luzokhula nge-120 bp ngemuva kokuthi i-adaptha iboshiwe. Uma ucezu lwe-DNA lukhula ngaphezu kwe-120 bp ngemuva kwe-adaptha ligation, kungahle kudalwe ukukhuliswa kwesiqeshana okungavamile kokukhulisa ngokweqile kwe-PCR. Ukunciphisa inani lemijikelezo ye-PCR kungavimbela isimo.

c) Usayizi ongajwayelekile wezingcezu zomtapo wolwazi we-DNA ngemuva kwe-adaptha ligation Ubude be-adaptha kule kithi ngu-60 bp. Lapho imikhawulo emibili yesiqeshana iboshelwe ku-adapters, ubude buzokhuphuka nge-120 bp kuphela. Uma usebenzisa i-adaptha ngaphandle kwaleyo enikezwe yile khithi, sicela uxhumane nomhlinzeki ngokunikezela ngemininingwane efanele njengobude be-adaptha. Sicela uqinisekise ukuthi ukuhamba komsebenzi nokuhlola kulandela izinyathelo ezichazwe kumanuwali.

d) Usayizi ongajwayelekile wocezwana lwe-DNA ngaphambi kwe-adaptha ligation Isizathu sale nkinga singadalwa yizimo zokuphendula ezingalungile ngesikhathi sokuhlukaniswa kwe-DNA. Izikhathi ezahlukahlukene zokuphendula kufanele zisetshenziselwe okokufaka okuhlukile kwe-DNA. Uma okokufaka kwe-DNA kungaphezu kwe-10 ng, sincoma ukukhetha isikhathi sokuphendula semizuzu eyi-12 njengesikhathi sokuqala sokwenza kahle, futhi usayizi wesiqeshana okhiqizwe ngalesi sikhathi ikakhulu usebangeni le-300-500 bp. Abasebenzisi bangakhuphula noma banciphise ubude bezingcezu ze-DNA ngamaminithi ama-2-4 ngokuya ngezidingo zabo zokwandisa izingcezwana ze-DNA ngosayizi odingekayo.

I-A-2

a) Isikhathi sokuhlukaniswa asilungiswanga Uma ngabe i-DNA ehlukanisiwe incane kakhulu noma inkulu kakhulu, sicela ubheke Izinkombandlela Zokukhethwa Kwesikhathi Sokuhlukaniswa okunikezwe kumyalo wokuthola isikhathi sokuphendula, bese usebenzisa leli phuzu lesikhathi njengokulawula, ngaphezu kwalokho usethe uhlelo lokuphendula ukwelula noma ukunciphisa i-3 min ukwenza ushintsho olunembile ngesikhathi sokuhlukaniswa.

A-3

Ukusatshalaliswa kosayizi okungajwayelekile kwe-DNA ngemuva kokwelashwa kokuhlukaniswa

a) Indlela engafanele yokuncibilikisa i-reagent, noma i-reagent ayixubeki ngokuphelele ngemuva kokuncibilika. Thaw i-5 × Fragmentation Enzyme Mix reagent eqhweni. Uma usucibilikile, hlanganisa i-reagent ngokulinganayo ngokucindezela ngobumnene phansi kwephubhu. Ungavimbi i-reagent!

b) Isampula yokufaka ye-DNA iqukethe i-EDTA noma okunye ukungcola Ukuchithwa komoya usawoti kanye nama-chelating agents esinyathelweni sokuhlanzwa kwe-DNA kubaluleke kakhulu empumelelweni yesilingo. Uma i-DNA ichithwa ku-1 × TE, sebenzisa indlela enikezwe emyalelweni ukwenza ukwahlukaniswa. Uma ukugxila kwe-EDTA kusixazululo kungaqiniseki, kunconywa ukuthi kuhlanzwe i-DNA bese kuyichithwa emanzini asuswe ukuphendula okulandelayo.

c) Ukulinganisa okungalungile kokuqala kwe-DNA Ubungako be-DNA ehlukanisiwe buhlobene kakhulu nenani lokufakwa kwe-DNA. Ngaphambi kokwelashwa kokuhlukaniswa, i-quantification enembile ye-DNA esebenzisa i-Qubit, iPicogreen nezinye izindlela kubalulekile ukuthola inani eliqondile le-DNA ohlelweni lokuphendula.

 d) Ukulungiswa kohlelo lokuphendula akulandeli imiyalo Ukulungiswa kwesistimu yokuphendula ehlukanisiwe kufanele kwenziwe eqhweni ngokuya ngemiyalelo. Ukuqinisekisa umphumela omuhle kakhulu, zonke izinto zokuphendula kufanele zibekwe eqhweni futhi nokulungiswa kohlelo lokuphendula kufanele kwenziwe ngemuva kokupholisa okuphelele. Ngemuva kokuthi amalungiselelo eseqediwe, sicela uflick noma ipayipi ukuxubana kahle. Musa i-vortex!

1. Indlela yokuxuba engafanele (i-vortex, i-oscillation enobudlova, njll.) Izodala ukusatshalaliswa okungajwayelekile kwezingcezu zelabhulali (njengoba kukhonjisiwe kumfanekiso olandelayo), ngaleyo ndlela kuthinte ikhwalithi yomtapo wolwazi. Ngakho-ke, lapho ulungiselela isisombululo se-Fragmentation Mix reaction, sicela ngomusa ubhebhe phezulu naphansi ukuxuba, noma sebenzisa umunwe wokuthwebula ukuze uxube futhi uxube ngokulinganayo. Qaphela ukuthi ungahlangani ne-vortex.

2. Kumele kusetshenziswe i-DNA emsulwa ekwakhiweni kwelabhulali

■ Ubuqotho be-DNA obuhle: Ibhendi ye-electrophoresis ingaphezu kwama-30 kb, ngaphandle komsila

■ OD260 / 230:> 1.5

■ OD260 / 280: 1.7-1.9

3. Inani lokufakwa kwe-DNA kumele linembe Kuphakanyiswa ukuthi kusetshenziswe izindlela zeQubit nePicoGreen ukulinganisa i-DNA, kuneNanodrop.

4. Okuqukethwe kwe-EDTA kusisombululo se-DNA kumele kunqunywe ukuthi i-EDTA inethonya elikhulu ekuphenduleni koqhekeko. Uma okuqukethwe kwe-EDTA kuphezulu, ukuhlanzwa kwe-DNA kudinga ukwenziwa ngaphambi kokuhlolwa okulandelayo.

5. Isixazululo sokuphendula kokuhlukaniswa kufanele silungiswe eqhweni Inqubo yokuqhekeka iyazwela ekushiseni kokuphendula nesikhathi (ikakhulukazi ngemuva kokungeza isithuthukisi). Ukuze uqinisekise ukunemba kwesikhathi sokuphendula, sicela ulungiselele uhlelo lokuphendula eqhweni.

6. Isikhathi sokuphendula kokuhlukaniswa kufanele sibe nenembile Isikhathi sokuphendula sesinyathelo sokuhlukaniswa sizothinta ngqo ubukhulu bemikhiqizo yesiqeshana, ngaleyo ndlela kuthinte ukusatshalaliswa kobukhulu bezingcezu ze-DNA kulabhulali.

1. Hlobo luni lwesampula olusebenza kule khithi?

Uhlobo lwesampula olusebenzayo lwale kit lungaba yi-RNA ephelele noma i-mRNA ehlanziwe ngobuqotho be-RNA. Uma i-RNA ephelele isetshenziselwa ukwakha umtapo wolwazi, kunconywa ukusebenzisa i-rRNA depletion kit (Cat # 4992363/4992364/4992391) ukususa i-rRNA kuqala.

2. Ngabe amasampula e-FFPE angasetshenziselwa ukwakha umtapo wolwazi ngale khithi?

I-mRNA kumasampuli e-FFPE izokwehliswa isilinganiselo esithile, ngobuqotho obungekuhle. Lapho usebenzisa le khithi ekwakhiweni kwelabhulali, kunconywa ukuthi ukwandise isikhathi sokuhlukaniswa (ukunciphisa isikhathi sokuhlukaniswa noma ukungasebenzi kokuhlukaniswa).

3. Usebenzisa isinyathelo sokukhetha usayizi esinikezwe kumanyuwali yomkhiqizo, yini engadala ukuthi ingxenye efakiwe ibonakale iphambuka kancane?

Ukukhethwa kosayizi kuzokwenziwa ngokuhambisana ngokuqinile nesinyathelo sokukhetha usayizi kule manuwali yomkhiqizo. Uma kukhona ukuchezuka, isizathu kungaba ukuthi ubuhlalu bukazibuthe abulinganiseli ekamelweni lokushisa noma abuxubene ngokuphelele, ipayipi alinembile noma uketshezi luhlala kuthiphu. Kunconywa ukusebenzisa amathiphu nge-adsorption ephansi yesilingo.

4. Ukukhethwa kwama-adaptha ekwakhiweni kwelabhulali

Ikhithi yokwakha umtapo wezincwadi ayiqukethe i-adapter reagent, futhi kunconywa ukusebenzisa le kit ndawonye ne-TIANSeq Single-Index Adapter (Illumina) (4992641/4992642/4992378).

5. I-QC yomtapo wolwazi

Ukutholwa kwenani lomtapo wolwazi: Kusetshenziswa i-Qubit ne-qPCR ukuthola ukucwiliswa kwenqwaba kanye nokuhlungwa kwe-molar komtapo wolwazi ngokulandelana. Ukusebenza kuhambisana ngokuqinile nemanyuwali yomkhiqizo. Ukugxila komtapo wolwazi kuzohlangabezana nezidingo zokulandelana kwe-NGS. Ukutholwa kwebanga lokusatshalaliswa kwelabhulali: Kusetshenziswa i-Agilent 2100 Bioanalyzer ukuthola ibanga lokusatshalaliswa kwelabhulali.

6. Ukukhethwa kwenombolo yomjikelezo wokukhulisa

Ngokuya ngemiyalo, inani lemijikelezo ye-PCR ingu-6-12, futhi nenombolo yemijikelezo ye-PCR edingekayo kufanele ikhethwe ngokuya ngokufakwa kwesampula. Kumitapo yolwazi enesivuno esikhulu, ukukhuliswa okungaphezulu kuvame ukwenzeka ngamazinga ahlukahlukene, okukhonjiswa ngenani eliphakeme kancane ngemuva kokuphakama kobubanzi obukhonjiwe ekutholeni i-Agilent 2100 Bioanalyzer, noma ukuminyana okutholakele kweQubit kungaphansi kune-qPCR. Ukukhulisa okumnene ngaphezu kwesimo kuyinto ejwayelekile, engathinti ukulandelana kwelabhulali nokuhlaziywa kwedatha okulandelayo.

7. Ama-spikes avela kuphrofayili yokuthola ye-Agilent 2100 Bioanalyzer

Ukuvela kwama-spikes ekutholakaleni kwe-Agilent 2100 Bioanalyzer kungenxa yokuhlukaniswa okungalingani kwamasampuli, lapho kuzoba khona izingcezu eziningi ngosayizi othize, futhi lokhu kuzocaca kakhulu ngemuva kokunothisa kwe-PCR. Kulokhu, kuphakanyiswa ukuthi kungenziwa ukukhetha usayizi, isb ukusetha isimo sokuhlukaniswa sibe ngu-94 ° C ngemizuzu eyi-15 efukiwe, lapho ukusatshalaliswa kwesiqeshana kuncane futhi kugxilwe, futhi ubungqingili bungathuthukiswa.