Formalin-Fixed Paraffin-Embedding (FFPE) is a common preparation for biopsy specimens. A tissue sample is first fixed in formaldehyde (formalin) to preserve the tissue proteins and structures by chemically cross-linking proteins and other amine-containing molecules. The sample is next embedded in a paraffin block, making it easier to cut thin sections to mount on a slide for microscopy. FFPE samples, sometimes archival ones, also offer a valuable source for molecular studies such as DNA sequencing and RNA expression analyses and can provide insight into disease progression and prognosis, with cancer and neurological diseases being common subjects. However, gene expression analysis of FFPE tissues is often hindered by poor RNA quality, resulting from oxidation, or chemical cross-linking and other modifications induced by the fixation and embedding process. Recently, methods have been established that offer more reliable gene expression analyses on poor quality samples, although few studies have aimed to compare the effectiveness of different RNA extraction and analysis workflows.
A recent publication in Technology in Cancer Research & Treatment by Manjunath et al. compared several FFPE RNA workflows for use in NanoString® nCounter®-based RNA profiling. NanoString® is a probe-based technology that allows direct counting of mRNA transcripts and represents a potential means for analyzing degraded samples. The authors first compared two different RNA extraction methods, the AllPrep™ DNA/RNA FFPE RNA extraction kit (Qiagen) and Biotium’s CELLDATA RNAstorm™ FFPE RNA Extraction Kit. Using archived FFPE samples from patients with either colorectal neoplasia and a previous history of inflammatory bowel disease (IBD), IBD alone, or normal tissues, the samples were processed according to the respective manufacturer protocols. The RNAstorm™ FFPE RNA Extraction Kit employs CAT5™ technology for catalytic reversal of cross-linking and enhances removal of formaldehyde-induced damage. Using spectrophotometric and bioanalyzer measurements to quantify isolated RNA, the authors determined that A260/A230 values were statistically different between the RNAstorm™ FFPE RNA kit and the AllPrep™ DNA/RNA FFPE kit (P< 0.0001), suggesting that the RNAstorm™ kit performs better at removing contaminants (Figure 1). Additionally, the RNAstorm™ kit resulted in a higher total RNA yield compared to the AllPrep™ kit (P=0.0266). They then performed a titration experiment to evaluate the impact of mRNA degradation and mRNA input levels on gene expression profiling using the NanoString® platform. They concluded that the RNAstorm™ FFPE RNA Extraction Kit is a superior means of RNA extraction from FFPE, that adult FFPE samples yielded higher RNA levels than pediatric samples, and that the NanoString® nCounter is a reliable platform for analyzing FFPE samples even when measures such as A260/A230 and DV200 were suboptimal.
Figure 1. The RNAstorm™ FFPE kit leads to higher total yields and higher purity RNA extractions. A nonparametric Mann–Whitney U test was used to assess the statistical significance between standard measurements performed on RNA samples isolated from FFPE pediatric biopsy samples using either the RNAstorm™ FFPE RNA kit (n=15), or AllPrep® DNA/RNA FFPE kit (n=20), kits. The RNAstorm™ FFPE RNA kit resulted in a higher total RNA yield when compared to the AllPrep® kit (P=0.0266). In addition, the A260/A230 values were found to be statistically different between the RNAstorm™ and the AllPrep® prepared samples (P<0.0001), suggesting that the RNAstorm™ FFPE RNA kit performs better at removing contaminants. While average A260/A230 values were not ideal using either extraction kit, it is known that variable tissue preservation can affect the quality of RNA from FFPE tissues and there is currently no consensus on the suitable lower limit of this ratio. No statistical differences were observed between the average concentration and A260/A280 values. Credit: Figure produced using data reported in Manjunath et al. https://doi.org/10.1177/15330338221129710 under the Creative Commons license.
Learn more about Biotium’s suite of RNA and DNA extraction kits, especially those focusing on extraction from FFPE tissues. We also carry a variety of high quality and affordable DNA and RNA quantification kits and reagents as well as other molecular biology products.
Full Citation
Manjunath, H. S., Al Khulaifi, M., Sidahmed, H., Ammar, A., Vadakekolathu, J., Rutella, S., et al. Gene Expression Profiling of FFPE Samples: A Titration Test. Technology in Cancer Research & Treatment, 21, 15330338221129710 (2022). https://doi.org/10.1177/15330338221129710
AllPrep is a trademark of QIAGEN; nCounter is a registrered trademark of NanoString Technologies, Inc.