Alaabta

Dherer buuxa mRNA Taxane-Nanopore

Taxanaha RNA wuxuu ahaa qalab qiimo leh oo loogu talagalay falanqaynta qoraalka qoraalka ah.Shaki la'aan, taxanaha akhris-gaaban ee dhaqanku wuxuu halkan ka gaadhay horumarro badan oo muhiim ah.Si kastaba ha ahaatee, waxay inta badan la kulmaa xaddidaadyo aqoonsi isoform oo dherer buuxa ah, qiyaasid, eexda PCR.

Taxanaha Nanopore waxa uu ka soocaa qaybaha kale ee isku xigxiga, taas oo ah in nucleotide-yada si toos ah loo akhriyo iyada oo aan la samayn DNA oo ay abuurto akhris dheer oo tobanaan kilobases ah.Tani waxay awood siinaysaa akhrin toos ah oo ka gudba qoraalada dhererka buuxa iyo la tacaalida caqabadaha daraasadaha heerka isoform-ka.

Platform：Nanopore PromethION

Maktabadda:cDNA-PCR

Nala soo xiriir

Faahfaahinta Adeegga

Natiijooyinka Demo

Kiis barasho

Faa'iidooyinka Adeegga

● Eexda taxanaha hoose

● Muujinaya unugyo cDNA ah oo dhererkoodu dhan yahay

● Xog yar ayaa loo baahan yahay si loo daboolo isla tirada qoraallada

● Aqoonsiga soofoomyada kala duwan ee hidde kasta

● Qiyaasta muujinta heerka isoform

Tilmaamaha Adeegga

Maktabadda

Platform

Soo-saarka xogta lagu taliyay (Gb)

Xakamaynta tayada

cDNA-PCR (Poly-A tayaysan)

Nanopore PromethION P48

6 Gb/ muunad (waxay kuxirantahay noocyada)

Saamiga dhererka buuxa ：70%

Celceliska dhibcaha tayada: Q10

Falanqaynta bioinformatics

●Habaynta xogta ceeriin

● Aqoonsiga qoraalka

● Kala-bax kaduwan

● Qiyaasta muujinta heerka hidda-sidaha iyo heerka isoform-ka

● Falanqaynta odhaah kala duwan

● Faahfaahinta shaqada iyo kobcinta (DEGs iyo DETs)

Tusaalaha Shuruudaha iyo Bixinta

Tusaalaha Shuruudaha:

Nucleotides:

Conc.(ng/μl)

Qadarka (μg)

daahirnimo

Daacadnimo

≥ 100

≥ 0.6

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Xaddidan ama aan lahayn borotiin ama wasakh DNA oo lagu muujiyey jel.

Dhirta: RIN≥7.0;

Xoolaha: RIN≥7.5;

5.0≥28S/18S≥1.0;

xaddidan ama aan lahayn sare u qaadis aasaasi ah

Nudaha: Miisaanka (qallalan): ≥1 g

* Unugyada ka yar 5 mg, waxaan kugula talineynaa inaad soo dirto muunada nudaha barafaysan (ku jira nitrogen dareere ah).

Ka joojinta unugga: Tirada unugyada = 3×10⁶- 1×10⁷

* Waxaan kugula talineynaa in la raro unugyada la qaboojiyey.Haddii unuggaasi uu ka yar yahay 5×10⁵, Tooshka barafaysan ee dareeraha nitrogen ayaa lagula talinayaa, taas oo doorbidaysa soo saarista yar yar.

Muunada dhiigga: Mugga≥1 ml

Tusaalaha keenista lagu taliyay

Weel: 2 ml oo tuubo sentrifuge ah (Faylada qasacadaysan laguma talinayo)

Tusaalaha calaamadaynta: Kooxda+ku celcelinta tusaale A1, A2, A3;B1, B2, B3......

Shixnadda: 2, Barafka qallalan: Muunadodu waxay u baahan yihiin in lagu soo raro bacaha oo lagu aaso baraf qallalan.

Tuubooyinka RNAstable: Muunada RNA waxaa lagu qallajin karaa tuubada xasilinta RNA (tusaale RNAstable®) waxaana lagu rari karaa heerkulka qolka.

Socodka Shaqada Adeegga

Nucleotides:

Tusaalaha keenista

Dhismaha maktabadda

Isku xigxiga

Falanqaynta xogta

Adeegyada iibka kadib

Socodka Shaqada Adeegga

Nudaha:

Naqshadaynta tijaabada

Tusaalaha keenista

soo saarista RNA

Dhismaha maktabadda

Isku xigxiga

Falanqaynta xogta

Adeegyada iibka kadib

Hore: Taxanaha mRNA dhererka buuxa -PacBio

Xiga: 10x Genomics Visium Spatial Transcriptome

1.Falanqaynta muujinta kala duwan -Dhulka Volcano

Falanqaynta muujinta kala duwan ayaa laga baaraandegi karaa labadaba heerka hidda-sidaha si loo aqoonsado hiddo-wadaha la soo bandhigay (DEGs) iyo heerka isoform si loo garto si ka duwan.

3 (1)

qoraalada la muujiyay (DETs)

2.Khariidadda kulaylka ee kooxaynta kala sarraysa

3.Aqoonsiga iyo kala saarista beddelka ah

Shan nooc oo ah dhacdooyinka kala qaybinta beddelka ah waxaa saadaalin kara Astalavista.

4.Aqoonsiga dhacdooyinka beddelka poly-adenylation (APA) iyo Motif ee 50 bp kor u kaca poly-A

Kiiska BMK

Aqoonsiga kala-duwanaanshaha beddelka ah iyo tirinta heerka isoform-ka ee nanopore isku xigxiga qoraalka qoraalka dhererka buuxa

La daabacay:Isgaarsiinta Dabeecada,2020

Istaraatiijiyada isku xigxiga:

Kooxaynta: 1. CLL-SF3B1(WT);2. CLL-SF3B1 (K700E beddelka);3. Unugyada B ee caadiga ah

Istaraatiijiyada isku xigxiga: MinION 2D taxane maktabadeed, PromethION 1D taxane maktabadeed;xogta akhris-gaaban ee isla muunado la mid ah

Madal isku xigxiga: Nanopore MinION;Nanopore PromethION;

Natiijooyinka muhiimka ah

1. Heerka Isoform-ka Beddelka Kala Qaybinta Aqoonsiga

Tixanaha akhriska dheer ayaa awood u siinaya aqoonsiga mutant SF3B1^K700E-la beddelo goobaha kala-goynta ee heerka isoform-ka.35 beddelka 3′SSs iyo 10 beddelka 5′SSs ayaa la ogaaday inay si weyn u kala duwan yihiin SF3B1^K700Eiyo SF3B1^WT.33 ka mid ah 35 ka beddelka waxa lagu ogaaday taxane-dheer oo la akhriyay.

2.Isoform-heer Alternative Splicing quantification

Muujinta haynta gudaha (IR) isoforms ee SF3B1^K700Eiyo SF3B1^WTayaa lagu qiyaasey iyadoo lagu salaynayo taxanaha nanopore, taasoo muujineysa hoos u dhac caalami ah oo ka jira soofoomyada IR ee SF3B1^K700E.

Tixraac

Tang AD, Soulette CM, Baren MJV, iyo al.Tilmaamaha qoraalka dhererka buuxa ee isbeddelka SF3B1 ee lymphocytic leukemia daba-dheeraada waxay muujineysaa hoos u dhaca introns-ka la hayo.Isgaarsiinta Dabeecadda.