RNeasy Plant Mini Kit from Qiagen

Qiagen’s RNeasy Plant Mini RNA Extraction Kit is a widely used and generally robust kit which yields high quality RNA from a multitude of plant samples. It is the kit of choice in our lab although we have tried at least three in-house protocols and two other kits from Peqlab and Invitrogen. The reason we have stuck with Qiagen’s kit is its consistency, locally offered price and quick turnaround of RNA samples. Peqlab and Invitrogen RNA extraction kits are also very similarly placed in my opinion, in terms of price and RNA quality, but in my hands Qiagen’s kit wins out because of ease of use.

I use the kit primarily for extracting total RNA from Arabidopsis thaliana leaves. In our lab, we also work on cell cultures from French bean and leaf or stem tissue from tobacco, and the kit works fine with these different sample types as well. The kit recommends using 25-75 mg of starting material and I would recommend sticking to the higher end of that guide, i.e. 55 to 65 mg of starting tissue. I also find that the kit doesn’t stress enough just how important it is to effectively homogenize the starting material while maintaining it in a frozen state. The best way to achieve this in my hands is to work in a 4°C cold room, keep the weighed leaf samples frozen in liquid nitrogen in a 2 ml eppendorf tube and crush with a plastic tip grinder vigorously. Importantly, I would soak the tip grinders and any other apparatus which comes in contact with solutions or the plant material in DEPC water and 0.5% bleach and then double autoclave to eliminate RNase activity.

Once the plant material is ready, it is imperative to work quickly with the samples. I try never to exceed working with sets of 4 or 6 different samples at a time, to ensure that each is treated consistently. Since the entire protocol takes just about an hour, reducing the number of different samples is well worth it to ensure higher RNA yield and quality. The kit recommends 2 minute incubations at 56°C immediately following the addition of lysis buffer, which I find are useful when working with tough stem tissue from tobacco, which is difficult to manually homogenize. I would recommend optimizing tissue homogenization by mechanical means instead of tweaking the kit. With Arabidopsis leaf tissue, vortexing during lysis is usually sufficient. Following tissue lysis, the samples are put through a pair of Qiagen propriety columns which shred the plant debris and bind the RNA, respectively. Around these columns are a series of washes which de-salt and clean the sample to result in clean and high integrity RNA. I usually opt for the double elution option that the kit recommends in order to increase RNA yield. I also do this with sterile RNase-free water as opposed to any kind of Tris buffer. My elution volumes are usually 80 ul. I then quantify the RNA in a Nanodrop. All in all, I’d say the RNeasy kit by Qiagen is a very efficient product for high quality RNA recovery.