Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

The purpose of the formalin fixation and paraffin embedding (FFPE) protocol is to preserve proteins for tests such as FISH or immunohistochemistry, rather than nucleic acid strands. As TMAs do not require RNA extraction and apply fluorescent detection tests such as FISH and ISH, they can make use of the long-term storage and abundance of samples acquired from FFPE samples. Although FF samples provide better quality samples for microarray analyses, microarray oligonucleotide probes have consistency in detections from both FFPE and FF specimens. FFPE samples are also commonly used in microarray gene expression analyses. Due to differing tissue characteristics between FFPE and FF samples, the yield of cDNA after reverse transcription and amplification from FFPE and FF samples may vary. Through matched FF and FFPE samples studies to evaluate the integrity and consistency of gene expression in FFPE samples, it has been shown that FFPE samples may be used in gene expression analysis. 80, 81 Similarly, FF samples are preferred for RNA-Seq experiments as they provide better quality data. Studies have shown that the FFPE/FF pairs demonstrate a high correlation when analyzing gene expression if the storage time of specimens is less than two years, meaning that reliable RNA can be extracted from FFPE samples in younger tissue blocks. 81 Since it only requires the Capture and Reporter probes to anneal to the template, with the Reporter probe depicting the gene of interest, the sample is prevented from accumulating errors through the reverse transcription step, allowing FFPE samples to be utilized by NanoString nCounter. It has been shown that an nCounter experiment has a high concordance (90%) rate with immunohistochemistry, demonstrating that it is reproducible. Although nCounter shows promise in using FFPE samples, it is limited to detecting 770 genes at once. This limitation is present no matter which sample type is chosen. 81 Due to source variation and intratumor heterogeneity, individual FFPE tissues can have varying RNA qualities. Therefore, to produce the best results from FFPE tissues, a standardized method of RNA extraction must be established. In a study comparing the Trizol, Qiagen RNeasy FFPE, and Arcturus PicoPure RNA Isolation kits for quantity and quality of RNA for microarray gene expression studies, an efficient method for RNA extraction, from the deparaffinization to microarray hybridization steps, was developed. The protocol includes an RNeasy FFPE kit with a modified deparaffinization protocol for RNA isolation from FFPE tissues and a combination of X3P Array Ovation and FFPE WTA System kit for amplification and hybridization. This workflow improves on FFPE tissues as a reliable alternative for FF samples. 80