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Comparison of RNA yield from small cell populations sorted by flow cytometry applying different isolation procedures
Author(s) -
Mack Elisabeth,
Neubauer Andreas,
Brendel Cornelia
Publication year - 2007
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20391
Subject(s) - rna , rna extraction , lysis , chromatography , lysis buffer , context (archaeology) , extraction (chemistry) , reagent , dna , biology , flow cytometry , yield (engineering) , microbiology and biotechnology , membrane , chemistry , gene , biochemistry , materials science , paleontology , metallurgy
Background: RNA from sorted cell populations is crucial in many instances. We therefore compared four current protocols for RNA isolation, with regard to mRNA yield and purity. Moreover, we examined the effects on RNA recovery caused by different storage reagents. Methods: Small populations of K562 cells or PMBC were sorted into the lysing reagent and subjected to RNA extraction, employing either phase separation extraction using an acidic guanidinium–isothiocyanate reagent (TriFast™ reagent), the silica‐gel membrane‐based spin‐column technology (RNeasy Mini‐/Micro‐Kit™), or the isolation via paramagnetic oligo(d)T‐beads (μMACS™). Cells designated for delayed RNA isolation were kept either in RNAlater™, Qiagen Buffer RLT™, TriFast™ or PrepProtect™, or simply frozen after pelleting from PBS. The mRNA yield was determined by quantitative RT‐PCR. Results: Performing unpaired two‐tailed t ‐tests revealed that RNA was extracted in significantly higher amounts using magnetic bead isolation. This method also allowed best discrimination of induced IL2 gene expression. In contrast, phase separation extraction showed the highest rate of failures. Intermediate storage reduced RNA yield. Contamination by genomic DNA was detected in several samples subjected to phase separation or silica‐gel membrane‐based spin‐column extraction. Conclusions: Our results reveal advantages and disadvantages of RNA isolation procedures for small numbers of sorted cells and, therefore, facilitate the decision for the most appropriate protocol in a particular experimental context. © 2007 International Society for Analytical Cytology