RNA isolation and quantitative PCR from HOPE- and formalin-fixed bovine lymph node tissues

Abstract

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 μg/ml total RNA) and the Trizol method (87–161.1 μg/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 μg/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) (4.05×10 −2 pg/100 ng total RNA using the commercial kit and 6.45×10 −2 pg/100 ng total RNA using Trizol) in comparison to formalin-fixed tissues (5.69×10 −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.