The Absolutely RNA Miniprep Kit by Stratagene is designed to purify high-quality total RNA from small samples of tissues and/or cultured cells. This kit is designed to replace the typical purification scheme of guanidinium thiocyanate /phenol (essentially Trizol types of reagents) which involves use of organics that need to be disposed of as hazardous waste. Purified RNA should be 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 as RNA isolation from tissues, adherent or suspension cells, or RNA clean-up. Essentially, cells are lysed and transferred to a microcentrifuge tube and vortexed vigorously. The supernatant is transferred into the prefilter spin filter and centrifuged (presumably aids in shearing of DNA and lysing of cells). The filtrate is collected and 70% ethanol is added. The mixture is then placed into the RNA binding cup and centrifuged whereby the RNA is retained in the binding cup. A low salt wash is performed followed by a DNase digestion and then finally a high salt wash. RNA is then eluted in elution buffer. The standard protocol is optimized for RNA purifications from 20-40 mg of tissue samples or 106-107 cells in culture. 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 (beta-mercaptoethanol is supplied). 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 repeatedly attempted to isolate RNA from cells grown in culture on numerous occasions. I tried the kit with multiple cell lines including SUPT1, HeLa, and 293 cells. On every occasion that I tried this kit, I did not get the quality of RNA that was stated in the protocols. I used a NanoDrop spectrophotometer to quantify my RNA samples and routinely got les than 1 ug of total RNA with A260/280 nm ratios of <1.8. I would recommend sticking with Trizol types of isolations for repeated and good quality yields. Yields using a Tri Reagent with the same number of cells would typically be ~20-40 ug. I spoke to tech services about the kit and they could not offer any suggestions on why the kit did not work. In fact, tech services gave me credit for the kit.