Gene expression analysis by real-time reverse transcription polymerase chain reaction: influence of tissue handling

Factors such as warm ischemia and time at room temperature before tissue treatment may influence the results of mRNA expression analyses on tissue specimens obtained during surgery. We evaluated the effect of these factors on RNA integrity and mRNA expression levels by incubating freshly obtained mouse liver tissue at 25 or 37 degrees C for periods of 0-4 h. Changes in the mRNA expression levels of seven genes, Tbp, Eef1a, Fos, Junb, Myc, Vegf, and Glut2, were determined by real-time reverse transcription-polymerase chain reaction. Incubation at 25 degrees C for up to 4 h only slightly altered (by a factor of less than 2) levels of mRNA for Tbp, Eef1a, Junb, Myc, Vegf, and Glut2. This result is consistent with limited RNA degradation at this temperature. Incubation at 37 degrees C strongly affected the levels of these mRNAs. Four hours of incubation at this temperature resulted in extensive RNA degradation, with mRNA levels falling to 1/10th those before incubation. When relative quantification was performed, i.e., quantification of the target gene transcripts in comparison to an endogenous housekeeping transcript (Tbp or Eef1a), the changes in mRNA levels were reduced to less than 2.5-fold. Fos behaved very differently from the other genes tested on incubation, with Fos mRNA levels increasing considerably following incubation at either 25 or 37 degrees C. Our data suggest that, with the exception of certain genes induced by tissue injury, relative quantification of mRNA, even on degraded RNA samples, can provide a reliable estimate of in vivo mRNA levels.