NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis
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Optimization of Recovery of RNA From Formalin-fixed, Paraffin-embedded Tissue

Author(s): Chung JY, Braunschweig T, Hewitt SM

Publication: Diagn Mol Pathol, 2006, Vol. 15, Page 229

PubMed ID: 17122651 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to assess the effects of alternative deparaffinization processing of archived formalin fixed paraffin embedded (FFPE) tissues in reference to RNA yield, quality, and stabiliy.

Conclusion of Paper

Deparaffinization and lysis of archived FFPE tissues at higher temperatures and longer durations (95 degrees C, 65 degrees C for 3 days, respectively) drastically improved the yield of recovered RNA (2-fold) without affecting quality or stability when stored at -20 degrees C. The high temperature incubations require the use of an aqueous dewaxer.

Studies

  1. Study Purpose

    The purpose of this paper was to optimize deparaffinization and lysis conditions (deparaffinization reagent, temperature, duration) for FFPE tissue sections for maximum yield of high quality RNA. Tissues examined included breast, colon, kidney, liver, lung, tonsil, melanoma, ovary, and prostate.

    Summary of Findings:

    RNA recovery from FFPE tissue increased when deparaffinization was performed at a higher temperature (95 vs 80 degrees C) and lysis duration was extended (3 days vs 2 h) at 65 degrees C. Deparrafinization with a propriety aqeous dewaxer (AutoDewaxer) or xylene produced comparble results. In comparison to lysis for 24 h, incubation for 3 days increased RNA yield 170%. RNA quality, inferred by OD 260/280 ratios, was unaffected by processing modifications. The addition of alcohol rehydration steps did not affect the yield of recovered RNA.

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Deparaffinization Xylene
    Dewaxer
    Xylene + rehydration
    Dewaxer + rehydration
    80 degrees C
    95 degrees C
    Analyte Extraction and Purification Incubation duration/condition 2 h in Lysis Buffer at 65 degrees C
    16h in Lysis Buffer at 65 degrees C
    1 day
    2 day
    3 day
    4 day
    View more
  2. Study Purpose

    The purpose of this study was to evaluate RNA stability in samples extracted from FFPE tissues using modified deparaffinization conditions and stored at -20 degrees C for up to 60 days.

    Summary of Findings:

    RNA recovered from FFPE tissues using modified deparaffinization protocols that was stored at -20 degrees C exhibited stable yields and 260/280 OD ratios for up to 60 days when comared to RNA samples analyzed immediately.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 0 d
    15 d
    30 d
    60 d
    View more

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