Pregled bibliografske jedinice broj: 1142821
Comparison of RNA yield and purity from small cell numbers following TRI reagent, Direct-zol RNA Microprep and NucleoSpin RNA XS extraction
Comparison of RNA yield and purity from small cell numbers following TRI reagent, Direct-zol RNA Microprep and NucleoSpin RNA XS extraction // 45th FEBS Congress: Molecules of Life: Towards New Horizons (FEBS 2021)
Ljubljana, Slovenija, 2021. str. 332-332 doi:10.1002/2211-5463.13205 (poster, međunarodna recenzija, sažetak, znanstveni)
CROSBI ID: 1142821 Za ispravke kontaktirajte CROSBI podršku putem web obrasca
Naslov
Comparison of RNA yield and purity from small
cell numbers following TRI reagent, Direct-zol
RNA Microprep and NucleoSpin RNA XS extraction
Autori
Jirouš, Maja ; Zidar, Ana ; Glavaš, Kristina ; Mihalj, Martina ; Štefanić, Mario ; Tokić, Stana
Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni
Skup
45th FEBS Congress: Molecules of Life: Towards New Horizons (FEBS 2021)
Mjesto i datum
Ljubljana, Slovenija, 03.07.2021. - 08.07.2021
Vrsta sudjelovanja
Poster
Vrsta recenzije
Međunarodna recenzija
Ključne riječi
RNA extraction ; RNA yield
Sažetak
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.
Izvorni jezik
Engleski
Znanstvena područja
Interdisciplinarne prirodne znanosti, Temeljne medicinske znanosti
POVEZANOST RADA
Projekti:
UIP-2019-04-3494 - NGS analiza transkriptoma MAIT i ydT limfocita: fenotip, funkcija i raznolikost TCR klonova u podlozi razvoja vulgarne psorijaze (NGSmyPHENOTIP) (Tokić, Stana, HRZZ - 2019-04) ( CroRIS)
Ustanove:
Klinički bolnički centar Osijek,
Medicinski fakultet, Osijek