RNeasy Plant Mini Kit From Qiagen

The RNeasy Plant Mini Kit is used for the isolation of RNA from plant or filamentous fungi; isolated RNA can then be used in several downstream applications, such as reverse transcriptase PCR, poly A+ RNA selection, differential display, Northern blotting and cDNA synthesis. To begin, plant samples are ground in liquid nitrogen, subsequently disrupted and lysed in a denaturing buffer. One has to choose between two different lysis buffers depending on the starting material (presence of some secondary metabolites can cause solidification of the sample). The samples are then centrifuged through a QIAshredder column to homogenize and shear genomic DNA. After ethanol addition to provide the appropriate binding conditions, the RNA (longer than 200 bases) is bound on a silica membrane and is, after three washing steps, eluted in water.

Isolating RNA from plant material can be a difficult and tedious job. Success depends upon many factors like plant species, amount of starting material and of course, the presence of RNases. Also, experience plays a mayor role, more so than with most other laboratory experiments. In our laboratory, we have experience in isolating RNA from plants for many years and we still don’t get satisfactory results every time. To get more consistent results, we started to use the RNeasy Plant Mini Kit from Qiagen. An added bonus is the absence of a phenol/chloroform extraction step in this kit.

In our study on volatile production in plants upon herbivory attack, we use the kit for isolating RNA from Arabidopsis and Brassica. The isolated RNA is then used in reverse transcriptase-PCR on genes of interest. By using this kit, our success rate has gone up, but the yield is about half the amount (up to 25 µg from Arabidopsis) we get using a conventional phenol/chloroform based protocol. Since we only use the RNA for reverse transcriptase PCR, this is no problem for us, unlike DNA contamination. When using the kit, DNA is always present in our samples (contrary to what the manual states), sometimes even when we use the extended on-column DNase protocol (available separately, cat. no. 79254). A prolonged incubation with the DNase solution (up to half an hour) usually solves this problem. We’ve been using this kit for some years now – in a practical course as well as in our research. Usually more then 90% of the students successfully isolate RNA of good quality, something absolutely impossible with our conventional method.

Although this kit certainly makes things easier, many pitfalls that can reduce the success rate still exist, so one might want to get help of an experienced user when using this kit for the first time. The protocol consists of many steps and most of them are critical. Among these are grinding (material should stay frozen), the right amount of starting material (too much clogs the column) and prevention of RNase contamination. A strange feature of the kit is the use of pink and lilac colors to distinguish between shredder and binding columns. These colors are very similar and may cause unnecessary errors.

To summarize, I think this kit is a great kit for isolating RNA from plants. The reproducibility is especially impressive. Of course, some training in RNA isolation remains necessary but otherwise it is possible to master the procedure very quickly. The need for an add-on to eliminate DNA contamination is a drawback though.