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qRT-PCR from FFPE

Monday 5 December 2011

Formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.

Gene expression analysis of formalin-fixed paraffin-embedded (FFPE) material by quantitative real-time polymerase chain reaction is a valuable tool for the retrospective analysis of clinical samples.

The degraded nature of RNA from FFPE tissue can lead to variation in detection of gene expression between samples, hence, it is necessary to select reference genes that can reflect this variation.

Normalization

Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. (19861891)

To identify suitable references for the normalization of target genes in FFPE breast tumors, expression of 10 potential references were measured in 10 endocrine therapy-naive, primary invasive breast tumors.

The determination of marker genes and gene clusters involved in disease pathogenesis is increasingly contingent on high-throughput methods of gene expression profiling. However, the concurrently increasing application of mRNA from formalin-fixed and paraffin-embedded (FFPE) tissue archives, as well as cell-type-specific approaches by laser-assisted microdissection, frequently results in very small and degraded quantities of RNA.

Therefore, a successful amplification of cell-type-specific mRNA targets from FFPE tissues becomes more and more essential.

Several commercial amplification kits on FFPE single cells are available.

The approach of target-specific cDNA amplification is notably appropriate for subsequent real-time polymerase chain reaction, as a constant decrease of CT values by 14 polymerase chain reaction cycles could be demonstrated. (18382351)

References

- 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

- Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue. Green TM, de Stricker K, Møller MB. Diagn Mol Pathol. 2009 Dec;18(4):243-9. PMID: 19861891

- Optimized protocol for gene expression analysis in formalin-fixed, paraffin-embedded tissue using real-time quantitative polymerase chain reaction. Votavova H, Forsterova K, Stritesky J, Velenska Z, Trneny M. Diagn Mol Pathol. 2009 Sep;18(3):176-82. PMID: 19704263

- Selection of reference genes for normalization of qRT-PCR data derived from FFPE breast tumors. Drury S, Anderson H, Dowsett M. Diagn Mol Pathol. 2009 Jun;18(2):103-7. PMID: 19430294

- Reliability and reproducibility of a RNA preamplification method for low-density array analysis from formalin-fixed paraffin-embedded breast cancer samples. Ciotti P, Garuti A, Ballestrero A, Cirmena G, Chiaramondia M, Baccini P, Bellone E, Mandich P. Diagn Mol Pathol. 2009 Jun;18(2):112-8. PMID: 19430292

- Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area. Baba K, Pathak S, Sviland L, Langeland N, Hoosen AA, Asjo B, Dyrhol-Riise AM, Mustafa T. Diagn Mol Pathol. 2008 Jun;17(2):112-7. PMID: 18382372

- Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR. Theophile K, Jonigk D, Kreipe H, Bock O. Diagn Mol Pathol. 2008 Jun;17(2):101-6. PMID: 18382351