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


METHODS
Sixteen archival formalin-fixed paraffin-embedded (FFPE) myocardial tissues were subjected to RNA extraction by Trizol, SDS, and RNeasy 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 RNeasy FFPE kit, as compared to 14/16 by Trizol and 8/10 by SDS methods. The expression of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was observed in RNA extracted by RNeasy FFPE kit and Trizol. High yield of RNA was obtained by RNeasy FFPE kit than Trizol (P = 0.002) and SDS(P = 0.012). Of the three methods, RNeasy 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 B5.


CONCLUSIONS
The Qiagen RNeasy FFPE kit resulted in significantly high reproducibility of RNA from FFPE myocardial tissues, which are suitable for amplification by Taq-Man Real-Time and RT-PCR. Thus, the results show that these FFPE tissues can be used for gene expression, pathogen detection, and epidemiological studies.
Department of Virology, Postgraduate Institute of Medical Education and Research Chandigarh, India.
Mirnalini
M
Sharma
Baijayantimala
B
Mishra
Vandana
Uma N
UN
Saikia
Ajay
A
Bahl
Radha K
RK
Ratho
Kewal K
KK
Talwar
D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D016679:Genome, Viral; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006321:Heart / Q000821:virology*; D006801:Humans; D009206:Myocardium / Q000737:chemistry*; D016612:Paraffin Embedding / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D018709:Statistics, Nonparametric
D005557:Formaldehyde; D012313:RNA; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)