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.