RNA isolation and quantitative PCR from HOPE- and formalin-fixed bovine lymph node tissues
The use of RNA extracted from 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 M.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 analysed 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 GAPDH, total RNA from HOPE fixed tissues showed higher levels of target mRNA (4.05x10-2 pg/100ng total RNA using the commercial kit and 6.45x10-2 pg/100ng total RNA using trizol) in comparison to formalin fixed tissues (5.69x10-4 pg/100ng 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.