NGS Sequencing and Microarrays
Archival formalin-fixed, paraffin-embedded (FFPE) tissues from clinical trials constitute an immense resource for retrospective biomarker discovery projects. Depending on the study design and the time of collection of the FFPE tissue, molecular profiling of the samples may provide a powerful approach to identification of biomarker sets that are useful for patient stratification, monitoring drug efficacy and toxicity, or even prognosis1. Furthermore FFPE tissue can be accumulated and stored for long periods at minimal cost due to slow rates of degradation of protein and nucleic acids contained in the paraffin blocks and the advantages of room temperature storage2.
During the formalin fixation process, RNA becomes rigid from the addition of monomethyl groups to the bases as well as cross-linkages which form between nucleic acids and proteins. The fixed RNA is susceptible to shearing which generally results in the degradation of mRNA species and produces fragments with an average size of 200 nt and reduced quality. Degradation issues have previously made molecular characterization of mRNA derived from FFPE samples challenging. Novel methods to overcome formalin crosslinking between nucleic acids and proteins, during RNA isolation have substantially improved the quality of total RNA and thus enabled gene expression analysis using Illumina second generation sequencing platform. ORB utilizes the Clontech SMART-SeqStranded Total RNA - Pico Input Mammalian Kit for long RNA sequencing from FFPE tissue. See ORB's long RNA sequencing page for further information about our services to interrogate mRNA using Illumina's sequencing technologies.
Unlike mRNA, microRNAs are not significantly affected by the formalin fixation process used in preparing FFPE tissue samples. To exploit the opportunity of using archived samples to detect changes in microRNA expression levels in various disease states, ORB offers microRNA profiling from archived FFPE tissue samples. We prepare RNA from as little as 4 sections (20 microns each) using Ambion's RecoverAll Total Nucleic Acid Isolation Kit for FFPE, and enrich the low molecular weight RNA in the samples. Expression of microRNA in isolated RNA samples may be undertaken using either small RNA sequencing or microRNA microarray hybiridzation. To learn more about either service please clik on the appropriate link below. may be Samples are then labeled using Genisphere's Flash Tag RNA Labeling Kit and hybridized to ORB mirBASE 19 multispecies microarrays (human, mouse, or rat). The resulting FFPE microRNA expression profiles correlates well with that of microRNA expression profiles from fresh tissues. Visit ORB's microRNA microarray page for more information about this service.
Small RNA Sequencing
- Graveel CR, Calderone HM, Westerhuis JJ, Winn ME, Sempere LF. Critical analysis of the potential for microRNA biomarkers in breast cancer management. Breast Cancer (Dove Med Press). 2015 Feb 23;7:59-79. doi: 10.2147/BCTT.S43799. Review. PubMed PMID: 25759599.
- Webster AF, Zumbo P, Fostel J, Gandara J, Hester SD, Recio L, Williams A, Wood CE, Yauk CL, Mason CE. Mining the Archives: A Cross-Platform Analysis of Gene Expression Profiles in Archival Formalin-Fixed Paraffin-Embedded Tissues. Toxicol Sci. 2015 Dec;148(2):460-72. doi: 10.1093/toxsci/kfv195. PubMed PMID 26361796