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 mechanisms against agents that may induce oxidative stress. It has been proposed that renal HO gene expression regulates important hemoprotein(s) such as cytochrome P450 and may be essential to maintain homeostasis in the kidney. Because accurate assessment of HO-1 mRNA in normal and disease states in kidney were not available due to the limited number of cells, we developed a system to quantitate human HO-1 mRNA in samples limited in cell number and/or mRNA copies. Total RNA from human kidney was used to establish this technique; it was reverse-transcribed and then amplified by polymerase chain reaction (PCR) in a tube also 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 proportional to the amount of sample RNA, and it is therefore possible to calculate the number of specific mRNA molecules. We have used this approach to quantitate the number of HO-1 mRNA molecules in adenocarcinoma cells. Results show that reverse transcription (RT)/PCR methods were able to determine the number of HO-1 mRNA copies in biopsy samples of human adenocarcinoma cells.
Goodman
A I
AI
Rockefeller University, New York, New York 10021, USA.
Choudhury
M
M
da Silva
J L
JL
Jiang
S
S
Abraham
N G
NG
D000230:Adenocarcinoma / Q000201:enzymology*; D006419:Heme Oxygenase (Decyclizing) / Q000032:analysis* / Q000235:genetics; D006801:Humans; D007680:Kidney Neoplasms / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis
D012333:RNA, Messenger; D006419:Heme Oxygenase (Decyclizing)