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Fig. 6 | BMC Molecular Biology

Fig. 6

From: Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples

Fig. 6

Comparing different unstained glomerular transection counts in eight cases of cohort II. Each case is depicted by one symbol (open and filled triangles, squares, circles, one asterisk and one upside down open triangle) in (a), (b), (c), (e) and (f). There was a significant difference in RNA concentration (a) comparing 60 and 150 as well as 150 and 300 unstained glomerular transections (termed as “glomeruli” for the sake of convenience; *p < 0.05; **p < 0.01; Wilcoxon matched-pairs signed rank test). There was no significant difference in the RNA A260/A280 ratio (b). Relative expression of ADAMTS13 (c) demonstrated a significant difference between 150 and 300 micro-dissected glomerular transections. The replacement of five Cq values of ADAMTS13 in 20 micro-dissected glomerular transections by Cq = 36 resulted in a relative expression of < 0.01 in those five cases (c). In groups with at least 60 glomerular transections the relative expression of ADAMTS13 displayed a lower inter-assay coefficient of variability compared to groups with 20 glomerular transections (d). The lowest inter-assay coefficient of variability for relative expression of ADAMTS13 was in groups with 150 and 300 glomerular transections (d). For comparison, relative expression of GAPDH and POLR2A is shown (e, f). In 150 micro-dissected glomerular transections, relative expression of ADAMTS13, GAPDH and POLR2A displayed the lowest interquartile range. There was a significant difference among groups with 60 and 300 glomerular transections for GAPDH (e) as well as 150 and 300 glomerular transections for POLR2A (f; *p < 0.05). Relative expression was calculated using PGK1 and PPIA as normalization factor

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