Thus, our model closely reflects the glomerular changes of acute antibody-mediated rejection in humans and of a special subset of human transplant glomerulopathy. The significance of alloimmune immune complex-type deposits in human transplants deserves further investigation.
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.
BackgroundGlomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartment-specific glomerular gene expression analysis of formalin-fixed paraffin-embedded renal biopsies could improve research applications. The major challenge for such studies is to obtain good-quality RNA from small amounts of starting material, as applicable for the analysis of glomerular compartments. In this work, we provide data and recommendations for an optimized workflow of glomerular mRNA analysis.ResultsWith a proper resolution of the camera and screen provided by the next generation of micro-dissection systems, we are able to separate parietal epithelial cells from glomerular tufts. Selected compartment-specific transcripts (WT1 and GLEPP1 for glomerular tuft as well as PAX2 for parietal epithelial cells) seem to be reliable discriminators for these micro-dissected glomerular substructures. Using the phenol–chloroform extraction and hemalaun-stained sections (2 µm), high amounts of Bowman’s capsule transections (> 300) reveal sufficient RNA concentrations (> 300 ng mRNA) for further analysis. For comparison, in unstained sections from a number of 60 glomerular transections upwards, a minimum amount of 157 ng mRNA with a reasonable mRNA purity [A260/A280 ratio of 1.5 (1.4/1.7) median (25th/75th percentiles)] was reversely transcribed into cDNA. Comparing the effect of input RNA (20, 60, 150 and 300 micro-dissected glomerular transections), transcript expression of POLR2A significantly correlated when 60 and 150 laser micro-dissected glomerular transections were used for analysis. There was a lower inter-assay coefficient of variability for ADAMTS13, when at least 60 glomerular transections were used. According to the algorithms of geNormPlus and NormFinder, PGK1 and PPIA are more stable glomerular reference transcripts compared to GUSB, GAPDH, POLR2A, RPLPO, TBP, B2M, ACTB, 18SrRNA and HMBS.ConclusionsOur approach implements compartment-specific glomerular mRNA expression analysis into research applications, even regarding glomerular substructures like parietal epithelial cells. We recommend using of at least 60 micro-dissected unstained glomerular or 300 hemalaun-stained Bowman’s capsule transections to obtain sufficient input mRNA for reproducible results. Hereby, the range of RNA concentrations in 60 micro-dissected glomeruli is low and appropriate normalization of Cq values using our suggested reference transcripts (PGK1 and PPIA) allows compensation with respect to different amounts of RNA purity and quantity.Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0103-x) contains supplementary material, which is available to authorized users.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.