Comparison of RNA yield from small cell populations sorted by flow cytometry applying different isolation procedures.
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 (microMACS). 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.
Mack
Elisabeth
E
Department of Hematology, Oncology and Immunology, University of Marburg, Baldingerstrasse, 35043 Marburg, Germany.
Neubauer
Andreas
A
Brendel
Cornelia
C
D002458:Cell Fractionation / Q000379:methods*; D002469:Cell Separation; D004247:DNA / Q000302:isolation & purification; D005434:Flow Cytometry; D006146:Guanidines; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017879:Isothiocyanates; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D008280:Magnetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020871:RNA Stability; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results; D058428:Silica Gel; D012822:Silicon Dioxide; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D006146:Guanidines; D007376:Interleukin-2; D017879:Isothiocyanates; D012333:RNA, Messenger; C054435:guanidine isothiocyanate; D058428:Silica Gel; D012822:Silicon Dioxide; D004247:DNA; D013755:Tetradecanoylphorbol Acetate