Improved Purification and Yields of RNA by RNeasy®

Many relatively rapid methods are presently available for isolation of total RNA from tissues or cultured cells, most of which are reported to yield RNA of sufficient purity for Northern blotting, cDNA synthesis, cloning and reverse transcription-polymerase chain reaction (RT-PCR). One such recent method is in the form of a commercial kit called RNeasy® (Qiagen, Chatsworth, CA, USA) and is based on the selective binding of RNA in crude guanidinium isothiocyanate (GIT) extracts to silica beads within microcentrifuge spin columns. In this method, cells are lysed with a buffered solution of GIT, lysate-mixed with an equal volume of 70% ethanol and then spun through a silica column by centrifugation at 10000× g for 20 s. Impurities are washed through the column by centrifugation once with a solution containing GIT and twice with a solution of 80% ethanol. RNA is then eluted with a relatively small volume (30–60 μL) of nuclease-free water. We find RNeasy to be a very economical and effective way of rapidly purifying multiple samples (12 samples/h) suitable for Northern blotting, RT-PCR and other common applications. However, RNeasy is suitable for relatively small amounts of RNA (<50 μg). In this report, we describe a simple and rapid addition to this procedure that results in a 3-fold to 60-fold increase of RNA yield, while significantly improving RNA purity. Furthermore, this additional step shortens the total hands-on time required for RNA purification by RNeasy when greater than 1.8 mL per sample of extract is processed, by reducing the number of additions of extract to the column (each following a centrifugation step) from ≥6 to only 1. This improvement of the RNeasy method consists of extracting cells or