qRT-PCR

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis.

Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis.

As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types.

One round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples.

The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

Variants

 qRT-PCR from FFPE

References

 Evaluation of quantitative rt-PCR using nonamplified and amplified RNA. Ferreira EN, Maschietto M, Silva SD, Brentani H, Carraro DM. Diagn Mol Pathol. 2010 Mar;19(1):45-53. PMID: 20186012

 Analytical performance of a qRT-PCR assay to detect guanylyl cyclase C in FFPE lymph nodes of patients with colon cancer. Beaulieu M, Desaulniers M, Bertrand N, Deschesnes RG, Beaudry G, Garon G, Haince JF, Houde M, Holzer TJ. Diagn Mol Pathol. 2010 Mar;19(1):20-7. PMID: 20186008