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Characterization of RNA in Cytologic Samples Preserved in a Methanol-Based Collection Solution.

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  • 1Viral Exanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

Molecular Diagnosis : a Journal Devoted to the Understanding of Human Disease Through the Clinical Application of Molecular Biology
|February 25, 1999
PubMed
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The ThinPrep collection solution effectively preserves RNA for molecular analysis. Intact RNA was recovered from preserved cells, enabling successful amplification of cellular and human papillomavirus (HPV) mRNA.

Area of Science:

  • Molecular Biology
  • Cytopathology
  • Virology

Background:

  • ThinPrep is a widely used fluid-based method for cytological specimen collection and processing.
  • The need for preserving RNA integrity in specimens for downstream molecular analysis is critical.
  • Assessing RNA preservation capabilities of collection media is essential for accurate diagnostic testing.

Purpose of the Study:

  • To evaluate the efficacy of the ThinPrep collection solution in preserving RNA for molecular analysis.
  • To determine if RNA extracted from ThinPrep preserved samples is suitable for reverse transcription polymerase chain reaction (RT-PCR).
  • To assess the preservation of both cellular and viral RNA, including human papillomavirus (HPV).

Main Methods:

  • Utilized cervical cancer cell lines and cord blood lymphocytes for experimental validation.

Related Experiment Videos

  • Tested various protocols for fixation, storage (24 hours at room temperature or 4°C), and RNA extraction.
  • Analyzed total RNA integrity using denaturing gel electrophoresis.
  • Employed RT-PCR to amplify cellular and viral messenger RNAs (mRNAs).

    • Main Results:

    • Intact 28S and 18S ribosomal RNA were observed in cells preserved for 24 hours under tested conditions.
    • Successful amplification of cellular and viral mRNAs was achieved from preserved samples using RT-PCR.
    • Human papillomavirus (HPV) mRNA was detectable even in samples with as low as 10% HPV-positive cells.
    • RNA preservation in clinical samples proved adequate for cellular mRNA RT-PCR.

    Conclusions:

    • The ThinPrep collection solution maintains the integrity of total RNA in both experimental and clinical samples.
    • RNA preserved in ThinPrep media is suitable for successful amplification of cellular and HPV mRNA via RT-PCR.
    • This finding supports the use of ThinPrep for molecular diagnostic applications requiring RNA analysis.