Absolutely RNA Miniprep Kit From Stratagene

The Absolutely RNA Miniprep Kit by Stratagene enables researchers to purify high-quality total RNA from small samples of tissues or cultured cells. The kit is also useful for RNA clean-up following enzymatic reactions such as in vitro transcription. Purified RNA is of sufficient quality for most downstream applications, including quantitative RT-PCR, Northern blotting and microarray hybridizations.

The Absolutely RNA Miniprep kit includes pre-filter spin cups, RNA binding spin filters, collection tubes, DNase I and all necessary buffers (except for ethanol) for 50 samples. Detailed instructions provide specific protocols for various applications, such RNA isolation from tissues, adherent or suspension cells, or RNA clean-up. The standard protocol is optimized for RNA purifications from 20-40 mg of tissue samples or 10⁶-10⁷ cells in culture. The modified protocol is optimized for smaller sample sizes (5-20 mg tissue or 10⁵-10⁶ cells). Lyophilized DNase should be reconstituted in the provided buffer and ethanol should be added to the wash buffers prior to the first use of the kit. The lysis buffer should be supplemented with beta-mercaptoethanol prior to each isolation. Homogenized tissue samples or cell lysates may be stored in the lysis buffer at -80ºC. The standard protocol includes at least 9 centrifugation steps and takes approximately 40 minutes (including 15-minute DNase treatment). The RNA is eluted in 60-200 ul of elution buffer with expected yield of 5-25 ug total RNA from 10⁶ cells.

I have been using the Absolutely RNA Miniprep kit for RNA isolation from in vitro cultured cells for the last four years. The kit’s appearance and packaging have been recently improved. A previous intermittent problem with the caps of the collection tubes, which sometimes did not close properly with the spin columns inserted, has been resolved. Using the Miniprep kit, I successfully isolated total RNA from human lung epithelial cell line A549, and several lines of human and mouse T lymphocytes and macrophages (Jurkat, DO11.10, EL4, THP-1, RAW.264). However, I obtained unacceptably low yields when I tried to isolate total RNA from a mouse T lymphocyte cell line: D10.G4.1. An attempt to troubleshoot did not increase the yields, therefore, I had to use a different kit.

In addition, I used the Absolutely RNA Miniprep Kit to isolate total RNA from small-size mouse tissue samples (lungs, liver, spleen). The RNA appeared to be of very good quality, but contained trace amounts of genomic DNA despite DNase treatment. I also noted that the samples should be weighed very carefully since it is easy to overload the columns. I found TRIZOL reagent more practical since it easily accommodates larger tissue samples.

The best part of the Absolutely RNA Miniprep kit is in its utility for post-enzymatic RNA clean-up. I successfully used it to isolate RNA following immunoprecipitation of ribonucleoprotein complexes and proteinase K digestion.