Ribonucleic acid extraction from archival formalin fixed paraffin embedded myocardial tissues for gene expression and pathogen detection.

Introduction Archival tissue samples preserved in formalin are a great source of treasure for biomedical research and diagnostics. Formalin, though is a good preservative, causes the modification of nucleic acid limiting the application of fixed tissues. The present study evaluated three methods of RNAextraction for constitutive gene expression and pathogen detection. Material and methods Sixteen archival formalin-fixed paraffin-embedded (FFPE) myocardial tissues were subjected to RNAextraction by Trizol, SDS, and RNeasy FFPEkit followed by RT-PCRand Taqman Real-Time PCRto study the expression of housekeeping genes. Results RNAwas extracted from all 16 myocardial tissues (100%) by RNeasy FFPEkit, as compared to 14/16 by Trizol and 8/10 by SDSmethods. The expression of Glyceraldehye-3-phosphate dehydrogenase (GAPDH)was observed in RNAextracted by RNeasy FFPEkit and Trizol. High yield of RNAwas obtained by RNeasy FFPEkit than Trizol (P similar to=similar to 0.002) and SDS(P similar to=similar to 0.012). Of the three methods, RNeasy FFPEkit was evaluated for Enterovirus RNAdetection in 16 other histopathologically confirmed FFPEtissues of dilated cardiomyopathy (DCM) cases and Enterovirus genome was detected in 4/16 (25%) FFPEtissues of DCMcases. The enteroviral sequences of the viral isolates revealed 99% homology with Human coxsackievirus B5. Conclusion The Qiagen RNeasy FFPEkit resulted in significantly high reproducibility of RNAfrom FFPEmyocardial tissues, which are suitable for amplification by Taq-Man Real-Time and RT-PCR. Thus, the results show that these FFPEtissues can be used for gene expression, pathogen detection, and epidemiological studies. J. Clin. Lab. Anal. 26:279-285, 2012. (c) 2012 Wiley Periodicals, Inc.