High Pure RNA Isolation Kit From Roche Applied Science

This kit is designed for rapid isolation of total RNA from different types of tissue and cultured cells. It especially useful for total RNA isolation from relatively small amount of cultural cells (10⁶), 200 - 500 ìl of human blood , 10⁸ yeast cells, 10⁹ bacteria (gram positive or gram negative) cells. I have also successfully isolated total RNA from as few as five Xenopus embryos.

The RNA extraction is environmentally friendly, does not require phenol or other hazardous solutions and therefore, can be performed very conveniently on the lab bench, without the need for a fume hood. To use this kit, cells are first lysed in lysis buffer (included) and the cell debris is removed by centrifugation. The lysate is then applied to the glass fiber fleece in a High Pure Spin Filter Tube. The RNA binds to the glass fleece in the High Pure Tube, while contaminating substances (salts, proteins and other cellular contaminants) are washed off of the column through two subsequent washes; the first with Wash buffer I and the second Wash buffer II. DNA is then digested with DNase I (right on the filter). 90 ìl DNase Incubation Buffer plus 10 ìl of reconstituted DNase I is applied to the filter; the recommended incubation is for 15 min at room temperature. In our experience, however, it is advisable to increase the incubation time to at least 25-30 minutes because sometimes when isolated RNAs were tested in PCR reactions, we saw genomic DNA contamination. Usually the prolonged incubation eliminated this problem. Following incubation, a series of brief wash-and-spin steps quickly removes the resulting DNA fragments. The purified RNA is then eluted in a small volume of low salt buffer. The manual recommends elution in 50-100 ìl. I have found that it is more convenient to work with more concentrated RNA, so I recommend eluting in a smaller volume, for example, 25-30 ìl. In order to ensure complete RNA elution, it advisable to add hot (60°C) elution buffer and incubate High Pure Spin Filter Tube at 37°C for 10 minutes.

In our laboratory, this kit was successfully used for total RNA isolation from different sources, including cultural fibroblasts and neuroblastoma cells, mouse spleen tissue and Xenopus embryos. For the embryos, we used the protocol for RNA isolation from cultural cells. This protocol is easy and straightforward.

In conclusion, High Pure RNA Isolation Kit is a convenient, reliable, easy to use product and gives excellent reproducibility. I highly recommend it.