TIANSeq RNA Frag / cDNA Synthesis Module

Parizvino, yepamusoro-yekupfuuridza matekinoroji tekinoroji inowanzoenderana nechizvarwa chinotevera kuteedzera tekinoroji. Sezvo kureba kwekuverenga kwechizvarwa chinotevera kurongedza tekinoroji kuri kushoma, isu tinofanirwa kupaza hwakazara hurefu kuteedzana kuita zvidimbu zvidiki maraibhurari kuteedzana. Zvinoenderana nezvinodiwa zvekusiyanisa kwakasiyana kuyedza, isu tinowanzo sarudza imwechete-yakapera kuteedzana kana kaviri-kupera kuteedzana. Parizvino zvidimbu zveDNA zvechizvarwa chinotevera kurongedza raibhurari zvinowanzo kugoverwa muhuwandu hwe200-800 bp.

a) DNA haina hutano uye ine inhibitors. Shandisa yepamusoro-soro yeDNA sampuli kudzivirira kudzivirirwa kweiyo enzyme chiitiko.

b) Huwandu hweDNA sampuro haina kukwana kana uchishandisa PCR-yemahara nzira kuvaka DNA raibhurari. Kana iko kuiswa kweiyo DNA yakapatsanurwa ikapfuura makumi mashanu ng, PCR-yemahara kufambiswa kwekufambisa inogona kuitirwa sarudzo panguva yekuvaka raibhurari. Kana iyo kopi nhamba yeraibhurari yakanyanya kuderera kuti inyatso kuenzaniswa, raibhurari yeDNA inogona kukwidziridzwa nePCR mushure meadapter ligation.

c) RNA kusvibiswa kunotungamira mukusarongeka kwekutanga kweDNA quantification RNA kusvibiswa kunogona kuvapo mukuchenesa kweiyo genomic DNA, izvo zvinogona kutungamira kune isiriyo DNA quantification uye kukwana kweDNA kurodha panguva yekuvaka raibhurari. RNA inogona kubviswa nekurapa neRNase.

A-1

a) Zvidimbu zvidiki (60 bp-120 bp) zvinowoneka zvidimbu zvidiki zvinowanzo gadziridza zvidimbu kana zvidimbu zvinoumbwa nemaadapter. Kunatswa pamwe neAgencourt AMPure XP magineti emabheji anogona kunyatso bvisa zvidimbu izvi zveadapter uye kuve nechokwadi chekuteedzera mhando.

b) Zvimedu zvakakura zvinoonekwa muraibhurari mushure mekukudziridzwa kwePCR Hukuru hweraibhurari yeDNA chidimbu ichawedzera ne120 bp mushure mekunge adapta yazara. Kana chidimbu cheDNA chichiwedzera neanopfuura makumi matanhatu bp mushure meiyo adapta ligation, inogona kukonzerwa neasina kujairika kudhizaini kuwedzera kwePCR kukwidziridzwa. Kuderedza huwandu hwePCR macircuit kunogona kudzivirira mamiriro acho.

c) Saizi isina kujairika yemaraibhurari eDNA zvidimbu mushure meadapter ligation Kureba kweadapter mune ino kit ndeye 60 bp. Kana iwo maviri magumo echimedu akakwenenzverwa kune maadapter, iyo urefu inongowedzera chete ne120 bp. Paunenge uchishandisa adapta kunze kweiyo yakapihwa nekiti iyi, ndapota nyorera mutengesi kuti ape ruzivo rwakakodzera senge urefu hweadapter. Ndokumbirawo muve nechokwadi chekuti kuyedza kufambiswa kwebasa uye mashandiro anoteedzera nhanho dzakatsanangurwa mubhuku.

d) Saizi isina kuenzana yeDNA chidimbu pamberi peadapter ligation Chikonzero cheichi chinetso chinogona kukonzerwa nemamiriro ezvinhu asiri iwo panguva yekuparadzaniswa kweDNA. Nguva dzakasiyana dzekuita dzinofanirwa kushandiswa kune dzakasiyana DNA yekuisa. Kana iyo yekuisa yeDNA ichidarika gumi ng, tinokurudzira kusarudza nguva yekuita ye12 min senguva yekutanga yekugadzirisa, uye saizi yezvimedu inogadzirwa panguva ino iri kunyanya pakati pe300-500 bp. Vashandisi vanogona kuwedzera kana kudzikisa kureba kwezvimedu zveDNA zve2-5 min zvinoenderana nezvinodiwa zvavo kuti vagadzirise zvidimbu zveDNA nehukuru hunodiwa.

A-2

a) Nguva yekuparadzanisa haina kukwidziridzwa Kana iyo DNA yakapatsanurwa iri diki kana yakakura kwazvo, ndapota tarisa kune maGwara eFragmentation Nguva Sarudzo yakapihwa mumurairidzo kuti uone nguva yekuita, uye shandisa nguva ino sekutonga, nekuwedzera kumisikidza reaction system yekuwedzera kana kupfupisa 3 min kuti inyatso kugadzirisa panguva yekuparadzaniswa nguva.

A-3

Kukura kusina kujairika kweDNA mushure mekuparadzaniswa kurapwa

a) Nzira isiriyo yekunyungudika reagent, kana reagent haina kusanganiswa zvachose mushure mekunyungudika. Thaw iyo 5 × Fragmentation Enzyme Sanganisa reagent pachando. Kana uchinge wanyungudika, sanganisa iyo reagent zvakaenzana nekunyorovesa pasi pacho pombi. Usa vortex iyo reagent!

b) Iyo yekuisa yeDNA sampuro ine EDTA kana zvimwe zvinosvibisa Kupera kwemunyu ions uye chelating vamiririri muDNA yekuchenesa nhanho inonyanya kukosha pakubudirira kwekuyedza. Kana DNA ikanyungudutswa mu1 × TE, shandisa nzira yakapihwa mumirairo yekuita kupatsanurwa. Kana iyo EDTA yekumisikidzwa mumhinduro isina chokwadi, zvinokurudzirwa kuchenesa iyo DNA uye nekuinyungudutsa mumvura yakasviba kuti iite zvinotevera.

c) Yakasarudzika yekutanga DNA quantification Hukuru hweDNA yakakamurwa hwakabatana zvakanyanya neuwandu hwekuisa kweDNA. Pamberi pekuparadzaniswa kurapwa, chaiyo quantification yeDNA uchishandisa Qubit, Picogreen uye dzimwe nzira dzakakosha kuti uone huwandu chaihwo hweDNA mune yekuita system.

 d) Kugadzirirwa kweiyo reaction system hakuteedzere kuraira Kugadzirira kwekupatsanurwa maitiro maitiro kunofanirwa kuitwa pachando zvakanyatsoenderana nemirairo. Kuti uve nechokwadi chekuita kwakanakisa, ese maitiro ekugadzirisa anofanirwa kuiswa pachando uye kugadzirirwa kwekuita system kunofanirwa kuitwa mushure mekutonhora kwakazara. Mushure mekugadzirira kwapera, ndapota flick kana pombi kusanganisa zvakakwana. Usatore vortex!

1. Nzira isiriyo yekuvhenganisa (vortex, chisimba oscillation, nezvimwewo) zvinokonzeresa kugoverwa zvisina kujairika kwezvimedu zvemaraibhurari (sezvakaratidzwa mumufananidzo unotevera), zvichikanganisa kunaka kweraibhurari. Naizvozvo, paunenge uchigadzirira iyo Fragmentation Sanganisa mhinduro mhinduro, ndapota nyoro pombi kumusoro uye pasi kusanganisa, kana kushandisa chigunwe kuti ubatise nekusanganisa zvakaenzana. Ive nekuchengera kuti usasanganise ne vortex.

2. Yakachena kuchena DNA inofanira kushandiswa pakuvaka raibhurari

Good Kunaka kweDNA: Bhandi re electrophoresis rinodarika makumi matatu kb, risina tailing

■ OD260 / 230:> 1.5

■ OD260 / 280: 1.7-1.9

3. Mari yekuisa yeDNA inofanira kunge iri yechokwadi Inokurudzirwa kushandisa nzira dzeQubit nePicoGreen kuyera DNA, pane kuita Nanodrop.

4. Izvo zviri mukati meEDTA muDNA mhinduro inofanirwa kutemerwa EDTA ine simba rakakura pakuita kupatsanurwa. Kana zvirimo zveEDTA zvakakwirira, kucheneswa kweDNA kunofanirwa kuitiswa pamberi peyedzo inotevera.

5. Iyo yekuparadzanisa mhinduro mhinduro inofanirwa kugadzirirwa pamusoro pechando Iyo yekuparadzanisa maitiro inoenderana neyakaitika tembiricha uye nguva (kunyanya mushure mekuwedzera yekuwedzera). Kuti uve nechokwadi chekuita nguva yekuita, ndapota gadzirira maitiro system pane chando.

6. Iyo yekuparadzanisa yekuita nguva inofanirwa kuve yakajeka Iyo yekuita nguva yekuparadzanisa nhanho inokanganisa zvakananga saizi yezvimedu zvezvimedu, nekudaro zvinokanganisa saizi kugoverwa kwezvidimbu zveDNA muraibhurari.

1. Ndeupi rudzi rwemuenzaniso unoshanda kune ino kit?

Iyo inoshanda sampuro mhando yeiyi kit inogona kuve yakazara RNA kana yakanatswa mRNA ine yakanaka RNA kutendeka. Kana yakazara RNA ichishandiswa kuvaka raibhurari, zvinokurudzirwa kushandisa rRNA depletion kit (Cat # 4992363/4992364/4992391) kubvisa rRNA kutanga.

2. Ko mienzaniso yeFFPE ingashandiswa kuvaka raibhurari neiyi kit?

Iyo mRNA muFFPE sampuli ichashatiswa kune imwe nhanho, iine hushoma hushingi kuvimbika. Paunenge uchishandisa kit iyi pakuvaka raibhurari, zvinokurudzirwa kuti uwedzere nguva yekuparadzanisa (kupfupisa nguva yekuparadzanisa kana kusaita kupatsanurwa).

3. Uchishandisa danho rekusarudza saizi rakapihwa mubhuku rezvigadzirwa, chii chingaite kuti chikamu chakaiswa chioneke kutsauka kudiki?

Saizi sarudzo ichaitwa zvine mutsindo zvichienderana nesaizi yekusarudza nhanho mune ino chigadzirwa bhuku. Kana paine kutsauka, chikonzero chingave chekuti iwo magineti madhiragi haana kuenzana kutembiricha yemukamuri kana asina kusanganiswa zvakakwana, iyo pipette haina kunyatsoita kana iyo mvura inosara iri mupipi. Zvinokurudzirwa kushandisa iwo matipi ane yakaderera adsorption yeiyo kuyedza.

4. Kusarudzwa kwemaadapter mukuvakwa kwemaraibhurari

Iyo yekuvaka raibhurari kit haina adapter reagent, uye zvinokurudzirwa kushandisa kit iyi pamwe neTIANSeq Imwe-Index Adapter (Illumina) (4992641/4992642/4992378).

5. QC yeraibhurari

Library yakawanda kuwonekwa Iko kuvhiyiwa kuri mukuenderana nebhuku rezvigadzirwa. Iko kusangana kweraibhurari kunowanzo sangana nezvinodiwa zve NGS kuteedzana. Kuonekwa kwenzvimbo yekuparadzira raibhurari: Uchishandisa Agilent 2100 Bioanalyzer kuona renji yekuparadzira raibhurari.

6. Sarudzo yekuwedzera manhamba enhamba

Zvinoenderana nemirairidzo, huwandu hwePCR masekonzi i6-12, uye huwandu hwePCR macircuit anodiwa anofanirwa kusarudzwa zvinoenderana neyakapihwa muyenzaniso. Mumaraibhurari ane goho repamusoro, pamusoro pekusimudzira zvinowanzoitika mumatanho akasiyanasiyana, ayo anoratidzwa nehuremu hukuru zvishoma mushure mehurefu hwenongedzo pakutsvaga kweAgilent 2100 Bioanalyzer, kana iyo yakatarwa ye Qubit yakaderera pane iya ye qPCR. Hunyoro pamusoro pekuwedzeredza chiitiko chinowanzoitika, chisingakanganise kuteedzana kwemaraibhurari uye ongororo yedhisheni inotevera.

7. Spikes inoonekwa mune yekuonekwa chimiro yeAgilent 2100 Bioanalyzer

Kuonekwa kwema spikes muAgilent 2100 Bioanalyzer kuonekwa kuri nekuda kwekusaenzana kwemasampuli, uko kuchazove nezvidimbu mune imwe saizi, uye izvi zvichave pachena mushure mekuwedzeredzwa kwePCR. Muchiitiko ichi, zvinokurudzirwa kusaita saizi sarudzo, kureva kuti isa mamiriro ekuparadzaniswa kuita 94 ° C kwemaminetsi gumi nemashanu akaiswa mukati, uko kugovewa kwezvimedu kuri kudiki uye kwakamisikidzwa, uye homogeneity inogona kuvandudzwa.