The FastRNA® Pro Blue kit is an easy and quick way to isolate total RNA from gram positive and gram negative bacteria. The kit includes a manual with a simplified procedure for experienced users and a detailed protocol for first time users. All the required solutions are provided in the kit with the exception of chloroform and ethanol. This kit is great for gram positive bacteria since it uses a lysis matrix to break into the thick peptidoglycan layer.
To begin, an overnight culture is refreshed and allowed to incubate until reaching an OD600 of 0.9 – 1.0. The bacteria are pelleted and 1ml of the RNApro™ solution is added; this solution is specifically designed to inactivate cellular RNases during lysis. The solution is then transferred to the kit-specific Lysing Matrix B tubes with a blue cap (hence the name: Pro Blue Kit). These Lysing Matrix B tubes contain beads that facilitate bacterial lysing using friction, heat and physical interaction. The protocol then says to put the tubes in the FastPrep® Instrument for vigorous shaking. I found using a standard bead beater worked well, so the proprietary model is not required. After spinning off the beads, chloroform is added to dissociate the nucleoproteins. After spinning, the upper phase is put into a fresh tube and absolute ethanol is added to precipitate the RNA. The RNA pellet is washed with 100% ethanol, air dried and resuspended in DEPC-treated water. The manual goes on to show how to determine concentration and integrity for new RNA researchers. There is also an extensive troubleshooting section. The kit states the average yield from 1010 cells is 50ug.
This procedure has been the easiest and most reliable of all the RNA isolation kits I have used. Since I isolate from gram positive bacteria, RNA is quite difficult to obtain without lengthy procedures and minimal final RNA yields. I did not get the 50ug RNA yield, but I was able to get a high enough average yield for RT-PCR and Northern analysis. In my experiments, I noticed a high DNA contamination in my preps. Therefore, I found that an additional DNase treatment for 1-2 hours at 37ºC was necessary for more reliable results from downstream procedures. The lysing matrix really helps improve RNA yield without causing RNA degradation as compared to other kits that use enzymatic lysing.
Overall, this kit has made RNA isolation from difficult bacteria much quicker and more reliable. I have tried multiple kits and procedures but this FastRNA® Pro Blue Kit works the best and gives the highest concentration of RNA. With an added DNase treatment, the RNA from this kit works well for downstream applications.
Katrina Stumpf
M.S. Student and Senior Lab Specialist
McGuire VA Medical Center
Division of Infectious Disease