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Related Experiment Videos

Brain-specific polyA- transcripts are detected in polyA+ RNA: do complex polyA- brain RNAs really exist?

B P Fung1, M H Brilliant, D M Chikaraishi

  • 1Neuroscience Program, Tufts University School of Medicine, Boston, Massachusetts 02111.

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
|March 1, 1991
PubMed
Summary
This summary is machine-generated.

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Researchers found that long polyadenylated (polyA+) RNAs can be inadvertently nicked during standard isolation, appearing as shorter polyadenylated-negative (polyA-) transcripts. This artifact can be missed by Northern blot analysis, impacting RNA integrity assessment.

Area of Science:

  • Molecular Biology
  • Neuroscience
  • Genomics

Background:

  • Brain-specific polyadenylated-negative (polyA-) RNAs were previously characterized by rarity, polysomal association, and postnatal expression from single-copy genes.
  • Two rat genomic clones, rg13 and rg100, initially appeared to fit these criteria for brain-specific polyA- RNAs.

Purpose of the Study:

  • To investigate whether low levels of rg13 and rg100 transcripts detected in polyadenylated (polyA+) RNA fractions could be artifacts.
  • To determine if these transcripts fractionating as polyA- might result from the nicking of polyA+ RNA during isolation.

Main Methods:

  • Sensitive nuclease protection assays were employed to detect low transcript levels.
  • RNA integrity was assessed using known polyA+ RNAs: tyrosine hydroxylase (2-kb mRNA) and sodium channel (9.5-kb RNA).

Related Experiment Videos

  • Various RNA isolation procedures were used, including LiCl-urea and guanidine thiocyanate/CsCl centrifugation, followed by oligo(dT) cellulose chromatography.

    • Main Results:

    • Low levels (3-10%) of rg13 and rg100 transcripts were detected in polyA+ RNA fractions.
    • While the shorter tyrosine hydroxylase mRNA consistently fractionated as polyA+, the majority of the longer sodium channel RNA fractionated as polyA-.
    • Northern blot analysis revealed intact 9.5-kb sodium channel RNA only in the polyA+ fraction, with the polyA- fraction showing a smear, indicating random cleavage.

    Conclusions:

    • Long messenger RNAs (mRNAs) may be susceptible to nicking during standard RNA isolation procedures.
    • This nicking can lead to the misclassification of polyA+ RNAs as polyA-.
    • Northern blot analysis may not detect this random cleavage, potentially leading to misinterpretation of RNA integrity and characteristics.