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High throughput transcript discovery via array based normalisation of RACE libraries
 
   
Contents

ABSTRACT

 
RACE (Rapid Amplification of cDNA Ends) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. Here, we describe a strategy that uses array hybridization to improve sampling efficiency of human transcripts. The products of the RACE reaction are hybridized onto tiling arrays, and the exons detected are used to delineate a series of RT-PCR reactions, through which the original RACE mixture is segregated into simpler RT-PCR reactions. These are independently cloned, and randomly selected clones are sequenced. This approach is superior to direct cloning and sequencing of RACE products: it specifically targets novel transcripts, and often results in overall normalization of transcript abundances. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of novel transcripts, and we investigate multiplexing it by pooling RACE reactions from multiple interrogated loci prior to hybridization.

METHODS

 


DATA

 

RESULTS

 

  • RACEarray discovery of transcript isoforms

    Following is a more detailed version of figure 2b from the main text (i.e. with Ensembl annotations and fully expanded EST and mRNA tracks).
    We also provide two extra detailed figures representing RT-PCR verification of novel RACEfrags in different index loci.
    See main text for legend.

    • Figure 2b (CHAF1B locus)

    • KCNJ15 locus

    • RT-PCR-verified connectivity between an intergenic 5' RACEfrag and its index locus, KCNJ15 (a.k.a. NM_170736).
      Ten novel sequences were discovered, of which seven connect the two RT-PCR primers used.

    • SFI1 locus

    • RT-PCR-confirmed chimeric transcript.
      A 5' RACEfrag, lying within an exon of DRG1, connects with its index exon in locus RP5-858B16.1, 80 kilobases away, on the same strand.
      Note the intervening locus (RP11-247I13.2) on the opposite strand.


PROGRAMS

 

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