SwePub
Sök i LIBRIS databas

  Utökad sökning

WFRF:(Mignardi Marco)
 

Sökning: WFRF:(Mignardi Marco) > Landegren Ulf > Profiling and genot...

Profiling and genotyping individual mRNA molecules through in situ sequencing of super rolling circle amplification products

Wu, Chenglin (författare)
Mignardi, Marco (författare)
chen, lei, 1985- (författare)
Uppsala universitet,Institutionen för immunologi, genetik och patologi,molecular tools
visa fler...
Landegren, Ulf (författare)
Nilsson, Mats (författare)
visa färre...
 (creator_code:org_t)
Engelska.
  • Annan publikation (övrigt vetenskapligt/konstnärligt)
Abstract Ämnesord
Stäng  
  • We have recently developed a technology for localized sequence library preparation with rolling-circle amplification (RCA) as an approach for in situ sequencing. This method involves generation of clonally amplified and specially confined substrates for next-generation sequencing within the preserved context of cells and tissues. Our approach combines padlock probing, RCA, and sequencing-by-ligation chemistry that can resolve expression profiles of sets of genes and mutations in tissues without loss of histological context. Like other fluorescence-based assays, it can be hindered by high level of background fluorescence. To achieve high signal-to-noise ratios, we now describe a method to boost the amplification generated by RCA of padlock probes in situ by super RCA (sRCA). In this technique, a second padlock probe is hybridized, ligated and amplified on the first RCA product for enhanced, localized amplification. We describe and compare different sRCA strategies where gap-fill ligation was showed to be most efficient. The sRCA products co-localize and have comparable sizes as RCA products but they display at least two fold higher signal intensity. This increase in signal to noise also proved to result in two folds increase in the number of sRCA products detected. By combining sRCA with in situ sequencing for highly multiplex detection in tissue a four-time increase was seen. In summary, we demonstrate that sRCA can significantly increase the performance of padlock-based in situ sequencing for gene expression profiling of tissue sections, enabling detection of low abundant transcripts and the analysis of also highly auto-fluorescent samples. 

Ämnesord

NATURVETENSKAP  -- Biologi -- Genetik (hsv//swe)
NATURAL SCIENCES  -- Biological Sciences -- Genetics (hsv//eng)

Nyckelord

in situ sequencing; super RCA (sRCA); tissue gene expression profiling
gap-fill padlock probe

Publikations- och innehållstyp

vet (ämneskategori)
ovr (ämneskategori)

Till lärosätets databas

Sök utanför SwePub

Kungliga biblioteket hanterar dina personuppgifter i enlighet med EU:s dataskyddsförordning (2018), GDPR. Läs mer om hur det funkar här.
Så här hanterar KB dina uppgifter vid användning av denna tjänst.

 
pil uppåt Stäng

Kopiera och spara länken för att återkomma till aktuell vy