| Popis: |
RNA isolation providing high yield and purity is crucial when a limited number of sorted cells is used as a starting material for downstream RNAseq analysis. Here, we have used small numbers (1E4, 5x1E4 and 1E5) of peripheral blood mononuclear cells (PBMCs) to compare average RNA yield and purity following Direct-zol RNA Microprep (DZ), NucleoSpin RNA XS (NS) and TRI Reagent (TR) extraction procedure. Three healthy volunteers were recruited for blood collection and PBMC isolation, using discontinuous Lymphoprep centrifugation. Countess II cell counter was used for number and viability assessments prior to cell aliquot preparation. RNA quantification was performed by DeNovix DS-11 Series with the use of QubitTM RNA HS Assay Kit for fluorometry. The A260/280 absorbance ratio, Lin’s correlation coefficient (r) and Bland- Altman (BA) statistics were applied to estimate sample purity and concordance between spectrophotometric (SF) and fluorometric (F) RNA measurements. Compared to the average F RNA values obtained across all TR sample groups (2.4 ± 1.8 ng/μL), higher average concentration was observed in both DZ (4.9 ± 3.9 ng/μL, P < 0.02, Wilcoxon signed-rank test) and NS (4.8 ± 3.2 ng/μL, P < 0.01) RNA samples. Judging by the A260/280 ratios, NS (1.9 ± 0.3), but not DZ RNA (1.4 ± 0.3) samples were of higher purity compared to their TR counterparts (1.5 ± 0.1, P < 0.01). Alternative SF and F quantification values were concordant in DZ (r = 0.98), but not NS (r = 0.04) or TR (r = 0.02) samples, with a considerable BA differences [pair diff, mean(bias)] noticed across DZ (-3.2, pbias = 0.18), NS (-10.9, pbias = 0.012) and particularly TR (BA = -433 ng/μL, pbias = 0.009) cohorts. Our data demonstrate ability of all tested protocols for successful RNA isolation from as low as 1E4 cells, with higher average yield and purity achieved via DZ and NS protocols. TR, however, seems to be less effective and less reliable in samples with small cell numbers, particularly when combined with SF RNA quantification. |
