Abstract
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.
Highlights
Glomeruli are excellent pre-determined natural structures for laser micro-dissection
We focused on two candidate reference transcripts (GAPDH and ribonucleic acid (RNA) polymerase isolated glomerular transections and (II) subunit A (POLR2A) [polymerase (RNA) II (DNA directed) polypeptide A)] and one target transcript [ADAMTS13] due to our interest in thrombotic microangiopathy
Patients and biopsies Overall, formalin-fixed paraffin-embedded (FFPE) native and transplant kidney biopsies from 24 patients were selected from the archive of the Institute of Pathology, Hannover Medical School and Institute of Pathology, Department of Nephropathology, Friedrich-Alexander University Erlangen-Nuremberg. (i) Nine native kidney biopsies with acute tubular injury (ATN) or interstitial inflammation without any glomerular disease were selected for the practicability of the micro-dissection of parietal epithelial cells (PECs, patient cohort I). (ii) In order to determine reference transcripts, we used an additional fifteen patients selected by a high number (> 30) of glomeruli per biopsy, including a wide range of native and transplant biopsies
Summary
Glomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartmentspecific 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. It has been shown that the majority of studies were performed with frozen tissue This limited experience with formalin-fixed paraffin-embedded (FFPE) studies is due to the fact that frozen tissue is preferred, which is most often available from animal studies. Often this is hardly an option for the large-scale analysis of human FFPE tissue from renal biopsies
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