The RNeasy Kit from Qiagen offers a fast and reliable procedure for purification of up to 100 µg total RNA from animal cells, animal tissue, yeast and bacteria. The kit contains all of the materials needed (except basic laboratory equipment) and avoids the use of phenol/chloroform extraction methods. It can also be used for RNA cleanup from crude RNA preparations and enzymatic reactions. For all applications, detailed protocols are included in the provided handbook.
The RNeasy procedure is easy to handle and straightforward. Cells are lysed and homogenized and total RNA is subsequently bound to an RNeasy spin column. After washing, high quality RNA can be eluted from the column. The maximum binding capacity per column is 100 µg total RNA. Molecules smaller than 200 nucleotides run through and will get lost during purification, thus, enrichment for mRNA is achieved. The purified RNA is ready for use in downstream applications such as RT-PCR, microarrays, differential display, and others. The kit should be stored at room temperature.
The RNeasy Kit is compatible with other RNA purification methods. We frequently use the kit to clean up crude (Trizol)- RNA preparations from human peripheral blood mononuclear cells (PBMCs). RNA cleanup is highly recommended for critical downstream applications such as RNA processing for mircoarrays. The purification procedure is very fast and allows additional on-column DNase digestion (not included in the kit) to remove any contaminating genomic DNA. Removal of remaining DNA is necessary only for very sensitive downstream applications. Depending on the expected amount, total RNA is eluted with 30 to 100 µl of provided DNase-free water. In our hands, yields and purity are excellent in about 90% of all performed purifications. The OD 260/280 ratios usually range between 2.08 and 2.19. Our main downstream application is RNA processing for microarray analyses. We never had any quantity or quality problems when cleaning up crude RNA preparations with Qiagen’s RNeasy Kit.