Ideal Solution for Finishing 5'-end cDNA Sequences, Identifying Alternate RNA Start-Sites, and Discovery of Tissue-Specific Promotors

Overview

Sure-RACE™ Multi-Tissue RACE Panels is a system for quickly completing sequence of the 5' ends of cDNAs. It is a PCR-based approach incorporating multiple "rapid amplification of cDNA ends" reactions that span some 24 individual human or mouse tissues. This simultaneous analysis of the 5' ends of a cDNA increases the chances of finding full-length sequences that would otherwise, in a single reaction, be often difficult to obtain. Sure-RACE panels also allows for the identification of alternate transcriptional start sites within the same tissue, in different tissues or at different developmental stages. Moreover, alternate RNA splicings in the 5' region of a transcript, which often lead to protein sequence variation, can be detected by this approach. Sure-RACE Multi-Tissue RACE Panels are supplied as double-stranded cDNA arrayed into 48-well PCR plates for convenient, direct amplification reactions.

 

 

How it works

First-strand cDNA is initially synthesized using an oligo(dT) primer and a reverse transcription reaction taking advantage of both the highly processive MMLV reverse transcriptase and the thermostable Tth DNA polymerase. The resulting highly representative cDNA is then "tailed" at its 3' end with terminal transferase and dCTP. Second-strand synthesis is followed using and adaptor-oligo(dG) to prime the C-tailed cDNA and Escherichia coli DNA polymerase I. The resulting double-stranded cDNA are then arrayed at two concentrations into 48-well PCR plates. Employing a gene-specific primer and an adaptor sequence primer, PCR is performed directly in the 48-well plate. A secondary PCR is then performed using a second "nested" gene-specific and adaptor sequence primer. The products of this reaction is then separated in an agarose gel for analysis.

 

Examples

1. To complete the 5'-end sequences of long and rare transcripts with extensive secondary structure.

The ataxia telangiectasia (ATM) gene encodes an ubiquitous 12-kb transcript with extensive conformations and heterogeneous 5' ends.

 

 

 

 

 

 

 

2. To discover alternate RNA start-sites derived from different tissue-specific transcriptional promotors.

The intercellular adhesion molecule-1 (ICAM-1) gene uses different promotors to initiate transcription.

 

 

 

 

 

 

 

 

3. To identify differentially-spliced transcripts that contain alternate 5' exons that are utilized in different tissues.

The fibroblast growth factor 1 (FGF-1) gene makes use of different 5' exons to regulate its expression.

 

 

 

 

 

 

 

 

 

 

Advantages

RAPID

  • perform PCR and run an agarose gel

VERSATILE

  • broad spectrum: scan 24 individual human or mouse tissues
  • high sensitivity: detect rare transcripts
  • simultaneous analysis of alternatively-spliced transcripts and alternate RNA start-sites

ECONOMICAL

  • each Sure-RACE contains 4 complete, identical 24-tissue panels for up to 4 separate experiments
  • Sure-RACE is about $24/tissue -- other RACE products can cost up to $390/tissue! -- Sure-RACE is the best value

 

 

 

 

 

 

 

 

 

Sure-RACE™ Multi-Tissue RACE Panel

Catalog Number

Human Sure-RACE™ Panel (4 Complete Panels*)

HRAA-101

Mouse Sure-RACE™ Panel (4 Complete Panels*)

MRAB-101

*Each Sure-RACE contains four identical 24-tissue, 48-well panels for up to four separate experiments.

Sure-RACE™ Multi-Tissue RACE Panel Applications Guide in Adobe .pdf format

      

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