QUANTITATIVE MEASUREMENT OF HEME OXYGENASE-1 IN THE HUMAN RENAL ADENOCARCINOMA

Heme oxygenase (HO-1) is the rate-limiting enzyme in heme catabolism. HO-1, a stress protein, has been suggested to be involved in defense m echanisms against agents that may induce oxidative stress. It has been proposed that renal HO gene expression regulates important hemoprotei n(s) such as cytochrome P450 and may be essential to maintain homeosta sis in the kidney. Because accurate assessment of HO-1 mRNA in normal and disease states in kidney were not available due to the limited num ber of cells, we developed a system to quantitate human HO-1 mRNA in s amples limited in cell number and/or mRNA copies. Total RNA from human kidney was used to establish this technique; it was reverse-transcrib ed and then amplified by polymerase chain reaction (PCR) in a tube als o containing an internal standard obtained by deleting 50 bp from the original human HO-1 gene. This allowed us to use the same primers for both the sample and internal standard. After amplification, templates were resolved by acrylamide gel electrophoresis and quantitated either by densitometry or radioactivity counted from the bands excised from the gel. When the internal standard is present in the reaction mixture , the ratio of amplified sample vs. the standard template is proportio nal to the amount of sample RNA, and it is therefore possible to calcu late the number of specific mRNA molecules. We have used this approach to quantitate the number of HO-1 mRNA molecules in adenocarcinoma cel ls. Results show that reverse transcription (RT)/PCR methods were able to determine the number of HO-1 mRNA copies in biopsy samples of huma n adenocarcinoma cells. (C) 1996 Wiley-Liss, Inc.