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Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization.

Author(s): Antonov J, Goldstein DR, Oberli A, Baltzer A, Pirotta M, Fleischmann A, Altermatt HJ, Jaggi R

Publication: Lab Invest, 2005, Vol. 85, Page 1040-50

PubMed ID: 15951835 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine whether degraded RNA extracted from formalin-fixed paraffin-embedded (FFPE) tumor specimens can be reliably analyzed by real-time quantitative RT-PCR (qRT-PCR) when normalized to stably expressed control genes.

Conclusion of Paper

Evidence of RNA degradation was present among extracts from FFPE specimens compared to RNAlater frozen case-matched controls. When normalized to control genes identified by the authors, relative expression of target genes were strongly correlated among specimens that were differentially preserved.

Studies

  1. Study Purpose

    The purpose of this study was to compare RNA quality and real-time qRT-PCR expression results among tumor specimens preserved in RNAlater and frozen with those fixed in formalin and paraffin embedded.

    Summary of Findings:

    RNA quality, as determined by 28S/18S ratios and fragment distribution, was superior in the RNAlater frozen specimens compared to case-matched FFPE specimens. Although differences in mean raw Ct values were observed between RNAlater frozen specimens and FFPE specimens, normalization to several control genes identified by the authors for their stable expression (SDHA, 18S, RPL0, HPRT1) resulted in a strong correlation in target gene relative expression between differentially preserved specimens.

    Biospecimens
    Preservative Types
    • Formalin
    • RNAlater
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    RNA Automated electrophoresis/Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    RNAlater
    Real-time qRT-PCR Specific Targeted nucleic acid SDHA
    18S
    RPL0
    HPRT1
    View more
  2. Study Purpose

    The purpose of this study was to determine if relative ER mRNA expression, determined by real-time qRT-PCR, and semi-quantitative ER protein expression levels, determined by immunohistochemistry, were in agreement among differentially preserved tumor specimens.

    Summary of Findings:

    ER protein levels among FFPE specimens and mRNA expression levels among FFPE and RNAlater frozen specimens were in good agreement for all 15 cases examined.

    Biospecimens
    Preservative Types
    • Formalin
    • RNAlater
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Protein Immunohistochemistry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    RNAlater
    View more

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