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Quantifying porphobilinogen deaminase mRNA in microdissected nephron segments by a modified RT-PCR
Dong Sun1, George Seki, Shu Uwatoko
1Department of Nephrology, Infectious Diseases, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
Background:
Quantifying mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR), although widely exercised, is still difficult.
Methods:
A modified quantitative RT-PCR in which genomic DNA was used as standard was developed. The quantity of mRNA was expressed as the ratio of the PCR product from cDNA and that from genomic DNA (CG ratio). Nephron distribution of porphobilinogen deaminase (PBGD) mRNA was examined in microdissected nephron segments using the method. The enzyme activity and mRNA quantity of PBGD also were measured in tissue homogenates.
Results:
Tubular segments expressed substantially more PBGD mRNA than glomeruli (expressed as CG ratios, 1.04 +/- 0.10 in glomeruli, 4.53 +/- 0.32 in PCT, 5.71 +/- 0.25 in PST, 5.13 +/- 0.52 in mTAL, 5.29 +/- 0.20 in cTAL, 4.05 +/- 0.35 in DCT, 2.88 +/- 0.25 in CCD, and 4.90 +/- 0.24 in OMCD). PBGD mRNA level in liver homogenate (3.17 +/- 0.36) was much higher than glomeruli but lower than most of the tubular segments. The enzyme activity in tissue homogenates correlated well with mRNA levels.
Conclusion:
The method reported here is simple and reliable, and especially suitable for quantitating specific mRNA amounts in minute tissue samples such as microdissected nephron segments.