Quantifying porphobilinogen deaminase mRNA in microdissected nephron segments by a modified RT-PCR.
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.


CONCLUSIONS
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.
Sun
Dong
D
Department of Nephrology, Infectious Diseases, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
Seki
George
G
Uwatoko
Shu
S
Nakao
Akihide
A
Goto
Atsuo
A
Fujita
Toshiro
T
Kimura
Satoshi
S
Taniguchi
Shigeo
S
D000199:Actins / Q000235:genetics; D000818:Animals; D017931:DNA Primers; D004210:Dissection; D015971:Gene Expression Regulation, Enzymologic; D011163:Hydroxymethylbilane Synthase / Q000235:genetics* / Q000378:metabolism; D008297:Male; D009399:Nephrons / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D017931:DNA Primers; D012333:RNA, Messenger; D011163:Hydroxymethylbilane Synthase