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Rare Transcripts Quantification by Strand-Specific RT-qPCR.

Ophélie Alyssa Martin1, Sandrine Le Noir1, Eric Pinaud2

  • 1Laboratoire Contrôle de la Réponse Immune B et des Lymphoproliférations (CRIBL), CNRS UMR 7276, INSERM U1262, Université de Limoges, Limoges, France.

Methods in Molecular Biology (Clifton, N.J.)
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PubMed
Summary

This study details a method for quantifying noncoding anti-sense transcripts at immunoglobulin loci during B lymphocyte development. This technique, using strand-specific reverse transcription, aids in understanding immunoglobulin gene accessibility and regulation.

Keywords:
Antisense transcriptsB lymphocytesMultiplex RT-qPCRRNA isolationStrand-specific

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Area of Science:

  • Immunology
  • Molecular Biology
  • Genetics

Background:

  • B lymphocyte development involves immunoglobulin (Ig) gene recombination, creating a diverse Ig repertoire.
  • Ig gene accessibility is regulated, partly through noncoding sterile transcripts, including anti-sense (AS) transcripts.
  • Quantifying these AS transcripts is crucial for understanding gene regulation.

Purpose of the Study:

  • To detail a method for quantifying strand-specific anti-sense transcripts at immunoglobulin loci.
  • To provide a protocol applicable to small cell numbers and adaptable to other cell types.
  • To emphasize the importance of experimental controls for accurate detection.

Main Methods:

  • Strand-specific reverse transcription (RT) of RNA isolated from cells.
  • Multiplex RT reaction and subsequent cDNA amplification.
  • Inclusion of specific experimental controls to validate strand-specificity and exclude false signals.

Main Results:

  • The described method allows for the quantification of low-abundant AS transcripts at Ig loci.
  • The protocol is optimized for RNA isolation from small cell populations.
  • Experimental controls are essential for reliable detection of strand-specific transcripts.

Conclusions:

  • The presented method enables precise quantification of AS transcripts, contributing to the understanding of Ig gene regulation.
  • This technique is valuable for studying gene accessibility in B cells and potentially other cell types.
  • Rigorous controls ensure the validity of detected strand-specific transcripts, advancing molecular biology research.