Open AccessRibonucleic Acid Extraction From Archival Formalin Fixed Paraffin Embedded Myocardial Tissues for Gene Expression and Pathogen Detection
Author(s) -
Sharma Mirnalini,
Mishra Baijayantimala,
Saikia Uma N.,
Bahl Ajay,
Ratho Radha K.,
Talwar Kewal K.
Publication year - 2012
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.21518
Subject(s) - trizol , rna , rna extraction , microbiology and biotechnology , taqman , housekeeping gene , nasba , nucleic acid , biology , gene expression , real time polymerase chain reaction , gene , chemistry , biochemistry
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 RNA extraction for constitutive gene expression and pathogen detection. Material and methods Sixteen archival formalin‐fixed paraffin‐embedded ( FFPE ) myocardial tissues were subjected to RNA extraction by Trizol, SDS , and RN easy FFPE kit followed by RT ‐ PCR and Taqman Real‐Time PCR to study the expression of housekeeping genes. Results RNA was extracted from all 16 myocardial tissues (100%) by RN easy FFPE kit, as compared to 14/16 by Trizol and 8/10 by SDS methods. The expression of Glyceraldehye‐3‐phosphate dehydrogenase ( GAPDH) was observed in RNA extracted by RN easy FFPE kit and Trizol. High yield of RNA was obtained by RN easy FFPE kit than Trizol ( P = 0.002) and SDS ( P = 0.012). Of the three methods, RN easy FFPE kit was evaluated for Enterovirus RNA detection in 16 other histopathologically confirmed FFPE tissues of dilated cardiomyopathy ( DCM ) cases and Enterovirus genome was detected in 4/16 (25%) FFPE tissues of DCM cases. The enteroviral sequences of the viral isolates revealed 99% homology with Human coxsackievirus B 5. Conclusion The Qiagen RN easy FFPE kit resulted in significantly high reproducibility of RNA from FFPE myocardial tissues, which are suitable for amplification by T aq‐ M an R eal‐ T ime and RT ‐ PCR . Thus, the results show that these FFPE tissues can be used for gene expression, pathogen detection, and epidemiological studies. J. Clin. Lab. Anal. 26:279‐285, 2012. © 2012 Wiley Periodicals, Inc.