Ambion’s ToTally RNA kit is ideal for isolating RNA from difficult to obtain samples such as human tissue. The procedure uses a guanidium thiocyanate denaturation solution but this reagent does not contain phenol unlike many commonly used RNA isolation solutions. Instead, there are separate phenol-chloroform-isoamyl alcohol and acid phenol chloroform extractions. The guanidium solution inhibits RNase activity and the sequential extractions remove protein, DNA, and contaminants. The kit includes a lithium chloride solution to purify the RNA even further if desired as well as RNase-free water with EDTA for the final resuspension.
I purchased the kit when a colleague recommended it for extracting RNA from postmortem human brain tissue. Since tissue from psychiatric patients is in limited supply, I did not want to take any chances with these precious samples. While single-step reagents work very well for rodent tissue or cultured cells, human RNA typically degrades partially during the postmortem interval and benefits from additional purification. Some of the precautions that best preserve RNA, such as immediately freezing the tissue, are not possible with autopsy samples. Some researchers who work with postmortem brain tissue use a single-step reagent such as Trizol to extract RNA and then further purify it on a column. However, this approach requires purchasing a variety of reagents and kits from different companies that are not necessarily optimized to work together.
Since I plan to use the RNA for quantitative RT-PCR, I treated the RNA with DNase after the precipitation. The RNase-free water used to resuspend the RNA is ideal for DNase treatment, since it contains a small amount of EDTA to inhibit RNase activity at elevated temperatures but not enough EDTA to affect the divalent cations in the DNase buffer.
One advantage is that the procedure can be scaled for any size sample by increasing the quantity of certain reagents and using a larger centrifuge. There are no columns to dictate the appropriate amount of tissue to use for each sample. In addition, the kit provides everything required for the isolation except isopropanol.
The clear disadvantage of this approach is that researchers expose themselves and their coworkers to phenol twice. For samples that provide high yields of intact RNA, a non-phenol-based approach or a single phenol extraction might be preferable, since it is quicker and safer. However, the ToTally RNA kit can be used for tissue, cultured cells, bacteria, or yeast.
As with any RNA isolation technique, certain aspects of the procedure will be left to the researcher. For tissue samples, adequate dissection and homogenization procedures are necessary to obtain suitable yields of intact RNA. This kit will reduce the number of contaminants that co-purify with the RNA, but it cannot undo damage caused by improper sample handling or inadequate tissue disruption. The majority of my samples were suitable for downstream analyses, but some were simply too degraded for even the best RNA isolation procedure.
Tara Lauriat
Graduate Student
Mount Sinai School of Medicine
New York, NY