There are several different viral RNA (vRNA) kits available for vRNA extraction. For example, the EZ1 Virus Mini Kit from Qiagen, working together with the BioRobot EZ1 workstation, is a very neat system and will totally free you from manual extraction. However, the maximum amount of starting plasma for the machine is 400 ul, and the minimum elution volume for vRNA is 60 ul. Considering the general recovery rate, it sometimes makes your final vRNA too dilute for downstream cDNA synthesis. This drawback will be extremely significant when you deal with low viral load samples. When searching the market, I was glad to find PureLink™ Viral RNA/DNA Kits from Invitrogen. They claimed to be compatible with a 500 ul initial input with final elution of the vRNA in as low as 10 ul of elution buffer. So it is worth a try. Actually, my first try with PureLink™ Viral RNA/DNA Kits was very successful, and the kit performed as promised.
Upon receiving my first kit, a yellow warning note attached on the top of the kit immediately attracted my attention: the lyophilized carrier RNA (cRNA) must be stored in -20ºC upon receiving. In my experience, in most cases the lyophilized cRNA are stable at room temperature. One year should not be a problem. I was not quite sure if it is necessary but followed their instruction. The good thing is their cRNA are <200 bp yeast tRNA and most of them are removed during the purification. It was good to know this because the cRNA present in the final extracted vRNA from other kits was always a concern when UV absorbance is used to quantify the vRNA.
After opening the PureLink™ Viral RNA/DNA Kit and dissolving the cRNA in RNase-free water, adding ethanol to the wash buffer and mixing a suitable amount of reconstituted cRNA with lysis buffer, I just went ahead to perform my vRNA purification. There was really nothing much to say; I simply followed the procedure. The kit is very well designed and easy to use. Two of my initial concerns turned out not important at all. One was the centrifugation after binding step. I thought it would be better for efficient binding to keep the centrifuging speed as low as possible. Well, there was no difference between 5,000 rpm and 10,000 rpm. Secondly, during elution, I tried to keep my tip at the center of the silica matrix to maximize the elution efficiency. Afterwards, I realized there is no need to do so because the tube containing the column is nearly cone shaped at the bottom. So even if you drop all the elution buffer on the wall of the tube, you just need tap the tube a little bit to make your buffer go right on top of the silica matrix. Great!
Overall, I used 400 ul of plasma sample to start, and eluted vRNA with 12 ul of water (I got 10 ul or so, which is expected). From my downstream assay, this kit indeed improved the total number of viral genomes to be analyzed by 5-6 fold due to its 6-times lower elution volume than the Qiagen BioRobot system. For processing 6 samples, it took me about 1 hr, which is as same as the BioRobot. However, with BioRobot, I can be totally free within the hour. So the benefit needs their payback.
Nonetheless, I have only used this kit to isolate viral RNA from HIV. The kit performance for other types of viruses needs to be further investigated, although they claim their lysis buffer is specifically formulated to allow lysis of different types of viral particles.