Identification of global gene expression patterns and specific gene expression levels are very common goals in many labs all over the world. Extraction of pure RNA from numerous types of cell and tissue samples which can then be used in downstream applications is of primary importance. But what if you do not have enough cells? Ambion has a kit that can remedy this situation. The RNAqueous-Micro Kit can be used for the purification of total RNA from very small, solid tissues samples (<10 mg), needle biopsies, microdissected cells, and small numbers of cultured cells (<500,000 cells).
The procedure uses guanidium thiocyanate to lyse the cells and rapidly inactivate ribonucleases. After guanidium thiocyanate treatment, the lysate is then mixed with ethanol. You use a silica-based filter that selectively binds RNA. The specialty of this silica filter is that it is very small, allowing elution of concentrated RNA in only ~10-20μl. You don’t need to further concentrate the RNA. Proteins, DNA and other contaminants are removed in three rapid washing steps. The procedure does not use phenol. The bound RNA is eluted with the pre-warmed elution solution. The kit supplies reagents for DNase-treatment. The DNase treatment step is performed after elution and is optional. After addition of the DNase reagent and 20 minute of incubation at 37 °C, you add a resin solution to inactivate the DNase and wait 2 minutes. Then you centrifuge the reaction and take the supernatant into a fresh tube
RNA isolation can be completed in 20 minutes following tissue disruption. One of the best things about the procedure is that it does not use phenol:chloroform extraction, protease digestion, or alcohol precipitation. This makes the RNAqueous-Micro kit ideal for processing many samples. The kit includes micro filter cartilage assemblies, micro elution tubes, wash solutions, lysis solution, LCM additive, DNase and its buffer, DNase reactivation reagent and elution solution for 50 RNA isolations.
We were looking for an RNA isolation kit that could be used with needle biopsy samples in our lab. After a deep search, we purchased the Ambion’s RNAqueous-Micro kit. One of the most attractive things about this kit is that the isolation procedure takes very little time. Because I use the samples for RT-PCR, I include the DNase treatment step. In total, obtaining RNA from four different tissue samples took 35-40 minutes. Also, I can say that the procedure is very easy. I obtained 10μl of RNA solution from each sample and the spectrophotometer results were satisfactory. The samples were also totally free of DNA.
My aim was to use the isolated RNA in an RT-PCR assay, but the resulting RNA is suitable for other applications, such as Northern blots, nuclease protection assays, in vitro translation and microarray analysis.
Taken together, the number of cells or the size of tissue sample will not restrict your research. I would recommend this kit for researchers who deal with small numbers of cells and want to do expression studies.
Molecular Biology and Genetics