Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies.
BACKGROUND
The appropriate choice of an internal reference is critical for quantitative RNA analysis. However, no comparison of frequently used "housekeeping" genes is available for renal biopsy studies.


METHODS
Microdissected biopsies from 165 patients, including a wide range of histopathologic diagnoses, were analyzed [immunoglobulin A (IgA) nephritis, membranous glomerulopathy, rapid progressive glomerulonephritis, minimal change disease, focal segmental glomerulosclerosis (FSGS), nephrosclerosis, diabetic nephropathy, interstitial nephritis, and controls]. Expression of three frequently used housekeeping genes [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and cyclophilin A] was examined by real-time reverse transcription-polymerase chain reaction (RT-PCR).


RESULTS
Absolute expression values of reference genes obtained from the renal biopsies were related to each other. In tubulointerstitial compartment, a positive correlation coefficient (r) of 0.96 was observed between 18S rRNA and cyclophilin A. However, a subset of samples showed lower expression levels for GAPDH in relation to 18S rRNA or cyclophilin A, resulting in a decrease to r = 0.77 and r = 0.73, respectively, consistent with considerable mRNA regulation of GAPDH. In glomerular samples, a comparable low correlation between GAPDH versus 18S rRNA (r = 0.75) was obtained.


CONCLUSIONS
Using a single housekeeper gene as reference for renal biopsy studies, differences in gene expression ratios may reflect regulation of the internal control rather than the mRNA under investigation. Relating the gene expression to several housekeepers in parallel (i.e., 18S rRNA and cyclophilin A) should result in robust expression data.
Schmid
Holger
H
Medizinische Poliklinik, Ludwig-Maximilians-University of Munich, Munich, Germany.
Cohen
Clemens D
CD
Henger
Anna
A
Irrgang
Sandra
S
Schlöndorff
Detlef
D
Kretzler
Matthias
M
D001706:Biopsy; D021984:Cyclophilin A / Q000235:genetics*; D004210:Dissection; D005786:Gene Expression Regulation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000378:metabolism* / Q000473:pathology*; D007674:Kidney Diseases / Q000378:metabolism* / Q000473:pathology*; D007678:Kidney Glomerulus / Q000378:metabolism / Q000473:pathology; D007684:Kidney Tubules / Q000378:metabolism / Q000473:pathology; D008866:Microsurgery; D012333:RNA, Messenger / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D021984:Cyclophilin A