Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable source of clinical material for research purposes. Consequently, is it important that researchers are able to utilize this resource efficiently, thus preventing any waste of precious samples. The Roche High Pure FFPE RNA Micro Kit is specifically designed to isolate total RNA from FFPE tissue, for downstream use in quantitative RT-PCR. Isolated RNA is free from inhibitors of RT-PCR, such as DNA, nucleases and cellular components. The kit boasts recovery of >80% with minimal RNA loss and a highly concentrated product. RNA can be isolated from 1-10 um sections, either cut as curls, or fixed to a microscope slide.
The first stage of the protocol involves a deparaffinization procedure which is carried out on sections individually. This procedure removes fixation reagents and involves xylene and ethanol washes. This step takes approximately 45-60 minutes and all reagents required must be user-supplied. The protocol is simple to follow, but care must be taken not to lose any of the tissue when removing the supernatant.
The second stage of the protocol is the RNA isolation stage. To each deparaffinized section, tissue lysis buffer is added along with SDS (user supplied). Tissues are then disrupted and homogenized by incubation with proteinase K or 3 hours at 55°C. A binding buffer is then added to samples and the remainder of the protocol is carried out using the supplied filters and collection tubes. At this point, if necessary, multiple preparations can be pooled. In the final stage, RNA is eluted from the glass fiber filters by centrifugation. This step is repeated in order to achieve maximal yield, producing approximately 20 ul of RNA product.
If photometric determination of RNA concentration is required, the eluted RNA must be centrifuged and the RNA supernatant removed without disturbing glass fibers at the bottom of the reaction tube. This step can result in a small amount of sample loss.
I have used the kit to isolate RNA from both FFPE tissue and formalin-fixed cell pellets. I found the kit to be successful in both cases, even when traditional methods such as Trizol extraction have failed. The RNA yield has been high, with a maximum concentration of 244 ng/ul. I routinely determine RNA concentration using a NanoDrop and in all cases RNA has been good quality with optimal 260/280 ratios. I use the isolated RNA in downstream quantitative RT-PCR and to date have not had any problem with any residual inhibitors of PCR.
High Pure FFPE RNA Micro Kit From Roche
High success rate with a high RNA yield from small tissue sections.
3 hour digestion step required, some sample loss if photometric determination of the RNA concentration is required.
The Bottom Line
An easy to follow protocol with a high success rate and high quality RNA product.