A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.
Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.
Del Prete
D
D
Division of Nephrology, Institute of Internal Medicine, University of Padova, Italy.
Forino
M
M
Gambaro
G
G
D'Angelo
A
A
Baggio
B
B
Anglani
F
F
D000199:Actins / Q000235:genetics; D001706:Biopsy; D004210:Dissection; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000378:metabolism* / Q000473:pathology*; D007700:Kinetics; D010446:Peptide Fragments / Q000235:genetics; D010865:Pilot Projects; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D000199:Actins; D010446:Peptide Fragments; D012333:RNA, Messenger; C068142:glyceraldehyde 3-phosphate dehydrogenase (304-313); D005987:Glyceraldehyde-3-Phosphate Dehydrogenases