Tuesday, May 11, 2010

Isolation of RNA from breast cancer FFPE tissues

In order to develop guidelines for clinical diagnostic tests using gene expression profiling based on qRT-PCR analyses of formalin-fixed, paraffin-embedded (FFPE) tissues, Sánchez-Navarro et al.* compared the performance of different normalization strategies in the correlation of quantitative data between fresh-frozen (FF) and FFPE tissues.  A significant challenge to expression analysis of FFPE samples is the substantial degradation of RNA extracted from these tissues, resulting in a shift in raw CT values compared to FF tissues. However, the authors demonstrate that proper normalization of the expression data can compensate of the effects of RNA degradation. The authors used the MasterPure™ RNA Purification Kit to isolate RNA from FFPE tissue slices and examined expression levels of reference and breast cancer prognosis–related genes using TaqMan® low-density arrays. Based on their analysis, they make recommendations for proper normalization strategies when using FFPE samples in qRT-PCR analysis. The authors conclude:

Nevertheless, careful selection of candidate biomarkers should be made: those genes that show no correlation between FF and FFPE should not be included in molecular tests for clinical use based in FFPE samples. Moreover, in order to guarantee reliable results in gene expression measurements, we strongly encourage performing preliminary studies, with the aim of discarding noncorrelated genes.

ResearchBlogging.orgSánchez-Navarro, I. et al. (2010). Comparison of gene expression profiling by reverse-transcription quantitative PCR between fresh frozen and formalin-fixed, paraffin-embedded breast cancer tissues BioTechniques, 48 (May 2010), 389-397 : 10.2144/000113388

6 comments:

wintermute said...

We are attempting to purify miRNA from FFPE slices, will this kit work?

epibio said...

Yes, the MasterPure RNA Kit will purify small RNAs, including microRNAs.

Biotech Desk said...

How does it compare with the Quick extract FFPE RNA kit of yours?

epibio said...

The MasterPure Kit provides highly purified RNA from FFPE samples, and consequently it is a longer procedure (approximately 1 hour). The RNA is suitable for a variety of applications.

In contrast, the QuickExtract FFPE RNA Kit is a "quick and dirty" isolation that takes about 30 minutes. The RNA is suitable for RT-PCR but not for other demanding applications.

anna said...

Hi, can the Masterpure been done on semen samples before. I am doing peal time PCR in goat semen

epibio said...

It will work fine for DNA; we haven't tested RNA purification.