RNAqueous Kit From Ambion

I worked in a pathology lab that was interested in isolating total RNA from human renal biopsy samples. These are small cores of tissues removed from patients for disease diagnosis following histochemical procedures. The remaining tissue from the needle biopsy samples was often archived by storage in -80ºC freezers. Thus pathologists never had a dearth of samples for molecular biology related experiments. However, the final amount of tissue that was available in each sample for RNA extraction was very miniscule and many a times would not be handled or prepared as recommended for nucleic acid extraction. I am sure there will be many pathologists out there who are hoping to find the right RNA extraction kit for their kind of starting material.

The RNAqueous Kit from Ambion is designed for the isolation of total RNA from small amounts of starting material. This kit follows a rapid, filter-based RNA isolation procedure. Thus this kit is ideal for processing large numbers of samples, as are generally used in clinical studies. This kit can be used to purify total RNA from many different animal and plant tissues, cultured cells, bacteria, yeast, and viral particles.

The amount of tissue that can be used in a single RNAqueous prep varies depending on the tissue source; generally, 1 to 75 mg of tissue or 100 to 10⁷ cultured cells is an appropriate amount of starting material. It is not advisable to use an excess of starting material as this will clog the filter column and reduce the final amount of RNA that is recovered. Cells or tissue are disrupted in a guanidinium thiocyanate solution which effectively lyses cells and inactivates endogenous ribonucleases. The lysate is then diluted with an ethanol solution and applied to an RNA-binding glass fiber filter. This is followed by three rapid washing steps to remove proteins, DNA and other contaminants. The bound RNA is then eluted in concentrated form. The entire procedure can be completed in 20 minutes, after tissue disruption. The procedure is suitable for processing many samples as it does not involve any phenol:chloroform extraction, protease digestion, or alcohol precipitation steps.

When I used the kit, Ambion suggested that increasing the volume of ethanol for diluting the lysate can help extract small molecular weight RNAs, like miRNAs. I have not tried this yet; however, their new protocol does not recommend using this kit for small molecular weight RNA species, which is a downside for this kit. It would be really useful if you could isolate all species of RNA from one experiment as these clinical samples are very precious. The company also has modified their RNA clean-up procedures. I have used the DNA-free DNase kit, which used to be part of the RNAqueous kit. It includes guaranteed RNase-free DNase I and reaction buffer, as well as a unique additive, the DNase Inactivation Reagent. After 20 min incubation at 37°C, DNase is inactivated quickly and easily by a 2 min incubation with the additive, without jeopardizing RNA in a heat treatment, and without organic solvents and alcohol precipitation. After the incubation, the RNA is spun down at maximum speed to pellet the inactivation reagent and remove the supernatant which will be the pure RNA. I was not very happy with this step as any carryover of the additive can interfere with the downstream applications.

Our objective was to use the RNA isolated from kidney biopsy samples for microarray hybridizations. The amount of RNA obtained using this kit was sufficient to perform amplification and labeling, and thus we were able to generate good data for our study. I am glad this kit worked well for us thereby helping us use the archival samples to generate valuable data that will aid in future development of biomarkers and therapy.