Glomeruli 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. With 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. Our 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.

中文翻译：

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从石蜡包埋的人肾活检样本中进行显微解剖的肾小球簇的mRNA分析的建议。

肾小球是用于激光显微解剖的出色的预定自然结构。福尔马林固定石蜡包埋的肾活检标本室特异性肾小球基因表达分析可以改善研究应用。此类研究的主要挑战是从少量起始材料中获得高质量的RNA，适用于肾小球区室的分析。在这项工作中，我们为优化肾小球mRNA分析的工作流程提供数据和建议。通过下一代显微解剖系统提供的摄像头和屏幕的适当分辨率，我们能够从肾小球簇中分离出壁层上皮细胞。所选的区室特异性转录本（用于肾小球簇的WT1和GLEPP1以及用于顶叶上皮细胞的PAX2）似乎是这些显微解剖的肾小球亚结构的可靠鉴别剂。使用苯酚-氯仿提取物和经半胱氨酸染色的切片（2 µm），大量鲍曼氏囊横切（> 300）显示出足够的RNA浓度（> 300 ng mRNA），可用于进一步分析。为了进行比较，在从60个以上肾小球横切向上的未染色切片中，反向转录了最小量的157 ng mRNA，具有合理的mRNA纯度[A260 / A280比为中值1.5（1.4 / 1.7）（第25/75个百分位数）]进入cDNA。比较输入RNA的效果（20、60、150和300个微解剖的肾小球横切），当使用60和150激光显微切割肾小球横断面进行分析时，POLR2A的转录本表达显着相关。当使用至少60个肾小球横切时，ADAMTS13的测定间变异系数较低。根据geNormPlus和NormFinder的算法，与GUSB，GAPDH，POLR2A，RPLPO，TBP，B2M，ACTB，18SrRNA和HMBS相比，PGK1和PPIA是更稳定的肾小球参考转录本。我们的方法甚至在涉及肾小球亚结构（如壁上皮细胞）的研究应用中，实现了针对小室的肾小球mRNA表达分析。我们建议至少使用60个微解剖的未染色的肾小球或300个经hemalaun染色的Bowman囊横切术，以获得足够的输入mRNA，以获得可重复的结果。特此，