UltraClean™ Tissue RNA Isolation Kit From MoBio Laboratories

The purification of high quality RNA from tissues and cells is essential to successfully perform downstream applications in molecular biology such as cloning and quantification of reverse-transcribed mRNAs through RT-PCR. Moreover, handling a lot of samples simultaneously requires a high-throughput method to ensure enough material from small tissue samples or small number of cells.

The UltraClean™ Tissue RNA Isolation Kit satisfies both of these characteristics enabling researchers to obtain high quality total RNA from small samples. It is based on RNA-binding spin filters and the kit contains all necessary buffers, solutions and collection tubes for 50 samples. DNase I enzyme, to digest DNA contamination, is not included; this is a weak point of the kit if you plan to carry out downstream reverse transcription reactions, but it is balanced by the low cost of the kit.

The standard protocol is optimized for RNA purifications from up to 30 mg of either fresh or frozen tissue samples or 10⁶ cells. The protocol is very simple to follow and is ideal for beginners too, since it is a sequence (6) of short (30 second) centrifugation steps alternating with the addition of a sequence of buffers (from TMR1 to TMR6). In my experience, best results were achieved without exceeding the starting amount suggested; this avoids DNA contamination and RNA degradation due to clogging of filters. If clogging occurs, it is useful to extend centrifugation time to 2-3 minutes as suggested in the troubleshooting section (which is very short, unfortunately). One further technical tip is to avoid to make sure to remove the DNA (which is clearly visible) from the spin filter before adding the TMR4 buffer; the DNA can be simply removed by aspiration; this greatly decreases the DNA contamination. The complete protocol takes approximately 50 minutes for up to 6 samples, from the homogenization step to the resuspension in RNase-free water (excluding the DNase treatment).

The RNA is eluted 50 µl of TMR6 buffer (RNase-free water) with expected average yields in the 10 to 25 µg range from 10⁶ cells or 10 to 70 µg from 30 mg of tissues (pancreas being on the low end and liver on the high end of that range).

I have used the UltraClean™ Tissue RNA Isolation Kit to extract total RNA from a variety of tissues from mammalian and non-mammalian vertebrates in order to study the expression of neuropeptides in the brain and gastrointestinal system through RT-PCR. Sometimes, I obtained low amounts of RNA, but the average result of the extractions is good.

I have also used the kit to obtain total RNA from cultured pancreatic beta islet cells from non obese diabetic mice (NOD). I found the kit effective for studying gene expression of the apoptotic pathways during the development of diabetes in the murine model.