Abstract
Small nucleolar RNAs (snoRNAs) have been used for normalization in glomerular microRNA (miRNA) quantification without confirmation of validity. Our aim was to identify glomerular reference miRNAs in IgA nephropathy. We compared miRNAs in human paraffin-embedded renal biopsies from patients with cellular-crescentic IgA-GN (n = 5; crescentic IgA-GN) and non-crescentic IgA-GN (n = 5; IgA-GN) to mild interstitial nephritis without glomerular abnormalities (controls, n = 5). Laser-microdissected glomeruli were used for expression profiling of 762 miRNAs by low-density TaqMan arrays (cards A and B). The comparison of different normalization methods (GeNormPlus, NormFinder, global mean and snoRNAs) in crescentic IgA-GN, IgA-GN and controls yielded similar results. However, levels of significance and the range of relative expression differed. In median, two normalization methods demonstrated similar results. GeNormPlus and NormFinder gave different top ranked reference miRNAs. Stability ranking for snoRNAs varied between cards A and B. In conclusion, we suggest the geometric mean of the most stable reference miRNAs found in GeNormPlus (miR-26b-5p), NormFinder (miR-28-5p) and snoRNAs (RNU44) as reference. It should be considered that significant differences could be missed using one particular normalization method. As a starting point for glomerular miRNA studies in IgA nephropathy we provide a library of miRNAs.
Highlights
Trace trace trace negative trace negative negative use as reference miRNAs highly questionable
First we performed a literature search in PubMed for articles published between January 2008 and December 2014, with the search terms “microRNA(s), micro-RNA(s) or miRNA(s)” and “kidney, renal, (IgA-) glomerulonephritis or (IgA-)nephropathy”
70 publications reported miRNA expression by RT-qPCR in renal tissue (18 times human, 39 times mouse, 13 times rat) and used small nuclear, nucleolar or ribosomal RNAs as well as mRNAs for normalization, namely snRNU6 (32 times), RNU48 (6 times), RNU87 (5 times), snoRNA202 (5 times), 5srRNA (5 times), 18srRNA (4 time), RNU6B (3 times) as well as others (GAPDH, RNU19, RNU44, RNU19, snoRNA 135, snoRNA 234) without confirming their validity for normalization. miR-193a and miR-16 were the only two reference miRNAs, which were properly identified as stably expressed in polycystic kidney disease and diabetic nephropathy, respectively[28,29]
Summary
Trace trace trace negative trace negative negative use as reference miRNAs highly questionable. The aim of the study was (i) to provide a comprehensive data set of glomerular miRNAs expression in native renal biopsies of IgA-GN on a cohort reflecting the full spectrum of glomerular tuft pathology (ii) to identify the most stably expressed miRNAs as a reference by proper normalization strategies (iii) to compare different normalization strategies for the identification of differentially expressed miRNAs in “active, cellular” crescentic IgA-GN vs IgA-GN vs controls. This provides basic information for further studies about the pathogenesis of IgA nephropathy especially to identify novel molecular markers for disease activity in urinary, serum and tissue diagnostics
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