RGD Reference Report - Quantifying porphobilinogen deaminase mRNA in microdissected nephron segments by a modified RT-PCR. - Rat Genome Database

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

Authors: Sun, Dong  Seki, George  Uwatoko, Shu  Nakao, Akihide  Goto, Atsuo  Fujita, Toshiro  Kimura, Satoshi  Taniguchi, Shigeo 
Citation: Sun D, etal., Kidney Int. 2002 Jan;61(1):336-41. doi: 10.1046/j.1523-1755.2002.00105.x.
RGD ID: 26923962
Pubmed: PMID:11786116   (View Abstract at PubMed)
DOI: DOI:10.1046/j.1523-1755.2002.00105.x   (Journal Full-text)


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.

Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
porphyrin-containing compound biosynthetic process  IDA 26923962uroporphyrin IRGD 
porphyrin-containing compound metabolic process  IDA 26923962Porphobilinogen RGD 

Objects Annotated

Genes (Rattus norvegicus)
Hmbs  (hydroxymethylbilane synthase)


Additional Information