A little while ago I was having technical difficulties obtaining total RNA from yeast cells for northern blot analysis. As usual, I was in a rush to get these experiments done, but I wasn’t sure which method to try next. Luckily, I was able to obtain a sample of Bio-Rad’s Aurum Total RNA Mini Kit at a product show at my institute.
The Aurum Total RNA mini kit is designed to purify total RNA from mammalian and yeast cells. Conveniently, this kit utilizes DNase I during the purification of total RNA to remove genomic DNA, not after the purification, hence, saving time. This kit can be used in two different ways: vacuum format or spin format. I used the spin format since the vacuum format requires an additional vacuum apparatus, which we don’t have in our laboratory. The spin format only requires the conventional 1.5 ml microcentrifuge tubes. The Bio-rad Total RNA Mini Kit comes with RNA binding columns, microcentrifuge tubes, lyophilized DNase I, lysis solution, wash solution, elution solution, and DNase dilution solution. In my case, when purifying total RNA from yeast cells, lyticase is required to help degrade the yeast cell wall and it is not supplied with the kit.
In my situation with working with yeast cells, this kit recommends 3.0 x 107 yeast cells per column. It is important not to use too many cells as there is the danger of clogging the column. With this amount of yeast culture, it is possible to get an average yield of 20-25 micrograms of total RNA using this kit.
The protocol for this kit is simple. Basically, you spin down your yeast cells when the OD-ml is about 3. Then, you add lyticase dissolved in lyticase dilution buffer, which is not included in the kit, to the cell pellet and re-suspend by pipetting up and down repeatedly and incubate for 10 minutes. The yeast cell/lyticase mixture is spun down and the supernatant is removed. Then, the cell pellet is resuspended with lysis solution, which is provided in the kit. 70% ethanol is added to the cell/lysis mixture and resuspended. Next, this lysate is added into the RNA binding column, which is inserted into the 2 ml capless tube, and spun quickly. The filtrate that passed through the column is discarded. Similarly, the RNA binding column is washed with low stringency wash solution, which is provided in the kit. Then, DNase I/DNase I dilution buffer, which is provided in the kit, is added to the column and allowed to incubate at room temperature. The columns are spun, and the DNase I/DNase I dilution buffer is discarded. The column is washed with high stringency wash solution and then with low stringency wash solution. Finally, the RNA is eluted from the RNA binding column by adding warm elution buffer, which is provided in the kit. Purifying total RNA with this kit is that easy. The protocol is very similar to many of the DNA purifying kits out there.
As always when dealing with RNA, one must take extra precautions to ensure that the reagents and apparatus are free of ribonucleases. Some of the reagents used in this kit (i.e. TE) will require treatment with DEPC. Also, once the RNase I is reconstituted, try to avoid repeated freeze/thaw cycles; hence, it is a good idea to aliquot out the RNase into multiple tubes. Overall, I am very pleased with this Aurum Total RNA Mini Kit by Bio-Rad, it makes purifying total RNA very simple and very efficient. I plan on purchasing this kit in the future when purifying RNA.
Hee Chul Lee
Graduate Student
Deptartment of Biochemistry
NYU School of Medicine