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条形 isibhengezo-03

Imikhiqizo

Ukulandelana kwe-mRNA yobude obugcwele-Nanopore

Ukulandelana kwe-RNA kube ithuluzi eliyigugu lokuhlaziya okuphelele kwe-transcriptome.Ngokungangabazeki, ukulandelana kwendabuko kokufunda okufushane kuzuze intuthuko eminingi ebalulekile lapha.Noma kunjalo, imvamisa ihlangabezana nemikhawulo ekuhlonzweni kobude obugcwele be-isoform, ukulinganisa, ukuchema kwe-PCR.

Ukulandelana kwe-Nanopore kuzihlukanisa kwezinye izinkundla zokulandelana, ngokuthi ama-nucleotide afundwa ngokuqondile ngaphandle kokuhlanganiswa kwe-DNA futhi akhiqize ukufunda isikhathi eside emashumini ama-kilobase.Lokhu kunika amandla okulotshiweyo okufundwayo okuqondile okuwela ubude obugcwele kanye nokubhekana nezinselele ezifundweni zeleveli ye-isoform.

Inkundla:I-Nanopore PromethION

Umtapowolwazi:cDNA-PCR


Imininingwane Yesevisi

Imiphumela yedemo

Ucwaningo lwesigameko

Izinzuzo Zesevisi

● Ukuchema kokulandelana okuphansi

● Ukwembula ama-molecule e-cDNA anobude obugcwele

● Idatha encane edingekayo ukuze kufakwe inombolo efanayo yokulotshiwe

● Ukukhonjwa kwama-isoform amaningi isakhi ngasinye

● Ukulinganisa kwenkulumo ezingeni le-isoform

Imininingwane Yesevisi

Umtapowolwazi

Inkundla

Isivuno sedatha esinconyiwe (Gb)

Ikhwalithi yokulawula

I-cDNA-PCR(I-Poly-A inothisiwe)

I-Nanopore PromethION P48

6 Gb/isampula (Kuya ngezinhlobo)

Isilinganiso sobude obugcwele>70%

Isilinganiso sekhwalithi yesikolo: Q10

 

I-Bioinformatics ihlaziya

Ukucubungula idatha eluhlaza

● Ukuhlonza umbhalo

● Okunye ukuhlanganisa

● Ukulinganisa kokuvezwa ezingeni lofuzo kanye nezinga le-isoform

● Ukuhlaziywa kwenkulumo okuhlukile

● Isichasiselo somsebenzi nokunothisa (ama-DEG nama-DET)

 

ubude obugcwele

Izidingo Zesampula Nokulethwa

Izimfuneko zesampula:

Ama-Nucleotides:

Conc.(ng/μl)

Inani (μg)

Ubumsulwa

Ubuqotho

≥ 100

≥ 0.6

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Amaprotheni anomkhawulo noma awekho nhlobo noma ukungcola kwe-DNA okuboniswa kujeli.

Okwezitshalo: RIN≥7.0;

Okwezilwane: RIN≥7.5;

5.0≥28S/18S≥1.0;

ukuphakama okulinganiselwe noma okungenakho

Izicubu: Isisindo(somile): ≥1 g

*Ngezicubu ezincane kuno-5 mg, sincoma ukuthi uthumele isampula yezicubu eziqandisiwe (ku-nitrogen ewuketshezi).

Ukumiswa kweseli: Isibalo samaseli = 3×106- 1 × 107

*Sincoma ukuthi uthumele i-frozen cell lysate.Esimeni lapho lelo seli liba lincane kuno-5×105, i-flash efriziwe ku-nitrogen eyi-liquid iyanconywa, okungcono kakhulu ekukhishweni okuncane.

Amasampula egazi: Ivolumu≥1 ml

Ukulethwa Kwesampula Okunconyiwe

Isitsha: 2 ml centrifuge tube (Tin foil ayinconywa)

Ukulebula okuyisampula: Ukuphindaphinda kweqembu+isb A1, A2, A3;B1, B2, B3....

Ukuthunyelwa: 2, Iqhwa elomile: Amasampula adinga ukupakishwa ezikhwameni futhi angcwatshwe eqhweni elomile.

  1. Amashubhu e-RNAstable: Amasampula e-RNA angomiswa ngeshubhu lokuzinzisa le-RNA(isb. i-RNAstable®) futhi athunyelwe ngegumbi lokushisa.

 

Ukugeleza Komsebenzi Wesevisi

Ama-Nucleotides:

ukulethwa kwesampula

Ukulethwa kwesampula

Ukulungiselela Umtapowolwazi

Ukwakhiwa komtapo wolwazi

Ukulandelanisa

Ukulandelanisa

Ukuhlaziywa kwedatha

Ukuhlaziywa kwedatha

Ngemva kokudayiswa Services

Amasevisi angemuva kokuthengisa

Ukugeleza Komsebenzi Wesevisi

Izicubu:

Isampula ye-QC

Idizayini yokuhlola

ukulethwa kwesampula

Ukulethwa kwesampula

Ukuhlolwa komshayeli

Ukukhishwa kwe-RNA

Ukulungiselela Umtapowolwazi

Ukwakhiwa komtapo wolwazi

Ukulandelanisa

Ukulandelanisa

Ukuhlaziywa kwedatha

Ukuhlaziywa kwedatha

Ngemva kokudayiswa Services

Amasevisi angemuva kokuthengisa


  • Okwedlule:
  • Olandelayo:

  • 1.Ukuhlaziywa kwesisho esihlukile -Isakhiwo seVolcano

    Ukuhlaziywa kwesisho esihlukile kungacutshungulwa kuzo zombili izinga lofuzo ukuze kukhonjwe izakhi zofuzo ezivezwa ngokuhlukile (ama-DEG) nakuleveli ye-isoform ukuze kukhonjwe ngokuhlukile.

     3(1)

    okulotshiweyo okuveziwe (DETs) 

    2.Imephu yokushisa yeqoqo le-Hierarchical

    4(1)

    3.Alternative splicing ukuhlonza kanye ngezigaba

    Izinhlobo ezinhlanu zezinye izehlakalo zokuhlanganisa zingabikezelwa yi-Astalavista.

    5(1)

    4.Ukuhlonza imicimbi ye-Alternative poly-adenylation (APA) kanye ne-Motif ku-50 bp phezulu nomfula we-poly-A

    6(1)

    Icala le-BMK

    Ukuhlonza okuhlukile kokuhlanganisa kanye nokulinganisa kwezinga le-isoform ngokulandelana kwe-nanopore yobude obugcwele be-transcriptome

    Kushicilelwe:Ezokuxhumana Ngemvelo, 2020

    Isu lokulandelanisa:

    Amaqembu: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E ukuguqulwa);3. Ama-B-cell ajwayelekile

    Isu lokulandelanisa: Ukulandelana kwelabhulali ye-Minion 2D, ukulandelana kwelabhulali ye-PromethION 1D;idatha yokufunda okufushane evela kumasampuli afanayo

    Inkundla yokulandelanisa: I-Nanopore MinION;I-Nanopore PromethION;

    Imiphumela esemqoka

    1.I-Isoform-level Alternative Splicing Identification

    Ukulandelana okufunde isikhathi eside kunika amandla ukukhonjwa kwe-SF3B1 eguquguqukayoK700E-altered splice sites at isoform-level.Amanye ama-3′SS angu-35 kanye nama-5′SS angu-10 atholwe ehlukaniswe ngokuphawulekayo phakathi kwe-SF3B1K700Ekanye ne-SF3B1WT.Okungu-33 kwezingu-35 okushintshiwe kusanda kutholwa ngokulandelana okufundwe isikhathi eside.

    2.Izinga le-Isoform-level Alternative Splicing quantification

    Ukuvezwa kwe-intron retention(IR) isoforms ku-SF3B1K700Ekanye ne-SF3B1WTbalinganiswa ngokusekelwe ekulandeleni kwe-nanopore, okuveza ukulawulwa phansi komhlaba wonke kwama-isoforms e-IR ku-SF3B1K700E.

    Ireferensi

    Tang AD , Soulette CM , Baren MJV , et al.Ukulingiswa kokulotshiweyo okugcwele kobude be-SF3B1 kuguquko lwe-lymphocytic leukemia engapheli kuveza ukulawulwa kwama-intron agciniwe[J].Ukuxhumana Kwemvelo.

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