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Directionality of noncoding human RNAs: how to avoid artifacts

Sivan Tzadok1, Yarden Caspin, Yafit Hachmo

  • 1Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

Analytical Biochemistry
|April 16, 2013
PubMed

Insights

Researchers discovered a common artifact in detecting long noncoding RNAs (lncRNAs) that falsely identified their presence. New methods using periodate treatment and high-temperature reverse transcription (RT) accurately detect these cancer-related RNAs.

Area of Science:

  • Epigenetics
  • Molecular Biology
  • Cancer Research

Background:

  • Epigenetic silencing of tumor suppressor and metastasis suppressor genes is common in human cancers.
  • Long noncoding RNAs (lncRNAs) are increasingly recognized as mediators of epigenetic silencing through chromatin modification.
  • Detecting promoter-spanning lncRNAs is crucial for understanding cancer development.

Purpose of the Study:

  • To investigate the presence and directionality of promoter-spanning lncRNAs in breast cancer cell lines.
  • To identify and overcome a prevalent artifact in lncRNA detection using reverse transcription polymerase chain reaction (RT-PCR).

Main Methods:

  • Screening of triple negative and Her2-overexpressing breast cancer cell lines for promoter-spanning transcripts.
  • Utilizing RT-PCR to detect transcriptional activity.
  • Implementing periodate treatment and high-temperature RT to eliminate a "no primer" artifact.

Main Results:

  • A high frequency of RT-PCR products was observed without exogenous primers, indicating a "no primer" artifact.
  • This artifact resulted from RT priming by promoter-associated transcripts, leading to false lncRNA identification.
  • Periodate treatment and/or high-temperature RT effectively eliminated the artifact, enabling accurate lncRNA detection.

Conclusions:

  • The study identifies and provides solutions for a significant artifact in lncRNA detection.
  • Accurate identification of promoter-spanning lncRNAs, such as those associated with BRMS1, RAR-β2, and CST6, is now possible.
  • These findings improve the reliability of studying lncRNAs involved in cancer epigenetics.

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