5 PRIME PerfectPure RNA Purification (formerly Gentra Systems' Versagene)

For researchers used to working with proteins or DNA, the prospect of isolating RNA can be a little frightening. We’ve all heard about the notoriously stable RNases and the precautions that must be taken to avoid them. Since simple autoclaving won’t permanently inactivate them, stronger measures such as diethylpyrocarbonate (DEPC) treatment of buffers and the baking of glassware overnight at high temperatures are required. Or, at least they used to be. Now, most disposable plasticware and premade buffers are available certified as DNase and RNase free. Also, solutions are available for the surface treatment of vessels and gel boxes to rid them of RNase contamination. All that’s left to worry about is the RNA isolation procedure itself since it is during cell and tissue lysis that cellular RNases are released.

Luckily, manufacturers of RNA isolation kits have taken most of the worry out of the isolation procedure as well. This is accomplished with premade lysis buffers containing strong chaotropes and protein denaturants to inactivate RNases. Since the lysis step of virtually all RNA isolation kits is pretty much the same, kit manufacturers have focused on simplifying the rest of the prep. In the past 6 months, I’ve used 3 different RNA purification kits. All gave similar results in terms of RNA quality but varied quite a bit in terms of convenience. The most recent kit, the Versagene RNA Cell Kit from Gentra Systems (now available as 5 PRIME PerfectPure RNA Cell Kit) has proven to be the most simple to use and results in very pure total RNA, based on 260/280 ratios and RT-PCR.

Unlike most other kits, all of the components of the 5 PRIME kit can be stored on the shelf. Also, all liquid components, of which there are only four, come ready to use. There is the option of adding a reducing agent (such as TCEP or beta-mercaptoethanol) to the lysis buffer for cells or tissues known to have high levels of RNase, but the lysis buffer alone is capable of inactivating RNases from most starting material. I used the kit to isolate total RNA from human blood lymphocytes and the results were great. After purifying the lymphocytes using density gradient centrifugation, I counted the cells and isolated RNA from about 5 million of them. The protocol is very simple: add the recommended volume of lysis buffer containing beta-mercaptoethanol to the cells and vortex for about 2 min. Transfer the lysate to a spin column and spin in a microcentrifuge for 1 min. Wash the bound RNA sequentially with Wash Buffers 1 and 2, transfer the column to a fresh tube and elute the RNA using the supplied elution buffer. Using this protocol, I recovered about 11 ug total RNA with 260/280 ratio of 1.91.

I tested the quality of the RNA by making cDNA and then performing PCR using primers that resulted in a 389 bp amplicon. The resulting band of the PCR product on a 1.5% agarose gel was indistinguishable from that obtained using lymphocyte RNA purified using a much more complicated prep involving lysis, phenol/chloroform extraction, and precipitation. In fact, the 260/280 ratio was better for the PerfectPure purified RNA (1.91 vs. 1.72).

Although the prep supposedly results in RNA essentially free of DNA, you can also perform on-column DNase treatment to be sure. This step is recommended when purifying RNA from whole blood but optional for other cells and tissues. In summary, the PerfectPure RNA Cell Kit is an excellent product. It is easy to use, fast, and results in RNA of excellent quality. Each spin column can handle the RNA from roughly 50 million cells. The quality and quantity of the RNA I recovered from only one-tenth this number indicates that the columns can accommodate a good range of cell numbers. Depending on the size of the kit purchased, the cost is around $3.50-3.95 per isolation. The leading spin column based RNA prep costs from about $4.10 to $4.85 per isolation.

At the time of this review the Versagene RNA Kit was commercialized by Gentra. Today it is available from 5 PRIME under the PerfectPure name. This review was edited to reflect this fact.