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Witchell J, Varshney D, Gajjar T, Wangoo A, Goyal M: RNA isolation and quantitative PCR from HOPE- and formalin-fixed bovine lymph node tissues. Pathol Res Pract. 2008;204(2):105-11. Epub 2007 Nov 5.
The use of ribonucleic acid (RNA) extracted from Hepes glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixed tissues in quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is fairly novel. We compared qRT-PCR analysis of formalin- and HOPE-fixed, paraffin-embedded lymph node tissues from Mycobacterium bovis-infected cattle by extracting total RNA using a commercial kit (Ambion) and a Trizol method. RNA extracted from HOPE-fixed tissues showed comparable quantities between the commercial kit (82.7-107.9 microg/ml total RNA) and the Trizol method (87-161.1 microg/ml total RNA), displaying a high degree of integrity when analyzed by electrophoresis. RNA extracted from formalin-fixed tissues using the commercial kit produced similar concentrations (80.6-145.7 microg/ml total RNA) in comparison to the HOPE tissue; however, the integrity was compromised. Extraction of RNA from the formalin-fixed tissues using Trizol was unsuccessful. Following qRT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total RNA from HOPE-fixed tissues showed higher levels of target messenger ribonucleic acid (mRNA) (4.05 x 10 (-2) pg/100 ng total RNA using the commercial kit and 6.45 x 10 (-2) pg/100 ng total RNA using Trizol) in comparison to formalin-fixed tissues (5.69 x 10 (-4) pg/100 ng total RNA). This could be attributed to RNA degradation by exposure to formalin fixation. In conclusion, the HOPE fixative proved to be a better source for RNA extraction from cattle lymph nodes and subsequent qRT-PCR. |
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