Good RNA preparation is a prerequisite to performing several experiments including cDNA synthesis, real-time quantitative PCR (qPCR) and promoter mapping. I have been using RNeasy Midi Kit for quite some time for the preparation of bacterial RNA. The kit, however, can also be used for RNA preparation from animal and plant cells. The contents of the kit are sufficient for 10 preparations. Another midi kit, enough for 50 preparations, is also available from the company.
The kit includes a lysis buffer that contains guanidine isothiocynate which not only lyses the cells, but also denatures almost all of the RNases. However, the solution does not contain any reducing agent and, therefore, 2-mercaptoethanol has to be added prior to use. Before lysis with the lysis buffer, cells have to be resuspended in TE buffer (Tris-EDTA) and treated with lysozyme. The TE buffer is not provided in the kit and has to be prepared separately. One should also ensure that this buffer is RNase-free. After lysis, the sample is spun at high speed to pellet down the debris. The supernatant with any floating precipitate is applied to the column. The column is spun at high speed and the flow through is discarded.
The wash buffer in the kit is provided as a concentrate, which should be diluted with ethanol prior to use, as instructed in the manual. After washing the column with wash buffer, the RNA is eluted in the RNase-free water available in the kit. The eluted RNA is almost always DNA-free and I have used this RNA directly for promoter mapping experiments.
For qPCR, where even residual amount of DNA can spoil the entire experiment, a DNase digestion step is required. The kit provides the advantage of on-column DNase digestion. RNase-free DNase is, however, not provided with the kit and has to be bought separately. The good part is that after on-column DNase digestion, a wash of the column removes all the DNase that was added. The purified RNA is thus free of any DNA or DNase. Phenol-chloroform extraction, which is usually carried out to remove DNase, is not required. I had to use DNase digestion for RNA used in qPCR experiments as I found that without this, the kit did not completely remove the all DNA, although DNA was not detected on an agarose gel.
Furthermore, the column also removes all RNA molecules smaller than 200 bases, such as 5S RNA and tRNA. Therefore, this kit cannot be used for isolation of any RNA smaller than 200 bases. After bacterial lysis is complete, the entire procedure takes nearly 20 minutes. Typical yields in my experiments with bacterial RNA preparations were 400-600 ug , which is more than sufficient for performing qPCR as well as promoter mapping with primer extension.
The kit comes with a handbook containing a detailed description of the RNA preparation as well as the necessary guidelines for working with RNA. It is advisable to read the guidelines before starting the experiment. All of the supplied columns and tubes are RNase free. Pipette tips should be DEPC treated prior to use. RNase inhibitor is not provided in the kit and, therefore, should be purchased separately.
One chemical that is not available in this particular kit is RNAlater, an RNA stabilizing reagent. Qiagen claims that adding this reagent to the lysate can stabilize RNA, which can then be stored for several months at –80ºC. I have not used this chemical yet and, therefore, cannot comment on its performance.
Vikas Jain
Research Scholar
Indian Institute of Science
Molecular Biophysics Unit