The PolyATract® mRNA Isolation System III is convenient, reliable, easy to use and gives very good quality mRNA. I highly recommend it.
For the microarrays I used as part of my PhD studies, I needed to isolate polyA mRNA. I tried several systems and found the PolyATract® mRNA Isolation System III the most convenient; it also produced a larger quantity of mRNA than the others I tested. The PolyATract® mRNA Isolation System III uses MagneSphere® technology to abolish need for oligo(dT) cellulose and its associated problems. This kit provides reagents sufficient to perform 15 mRNA isolations, starting with 1 mg or less total RNA per isolation. The polyA mRNA fraction can be isolated free of other nucleic acid contamination in approximately 45 minutes. The isolated mRNA is suitable for all molecular biology applications, including in vitro translation and cDNA synthesis.
The procedure itself consists of probe annealing when you have to add biotinylated-oligo(dT) probe and 13 µl of 20X SSC into the heated RNA sample then mix and cool at room temperature up to 10 minutes. Then you add your RNA mix to the prewashed magnetic particles, incubate them together, allowing mRNA-biotinilated oligo-dT hybrids bind to the streptavidin-associated magnetic particles. You then wash the particle-RNA hybrids from the total RNA and elute the purified mRNA.
I had to isolate mRNA from relatively small quantities (1 mg or less) of total RNA. I found that when I incubated the probe-RNA mixture at 37°C for 5 minutes and then cooled it for 5 minutes at room temperature, I got better results. Yields were also improved when the mixture was incubated with gentle rocking. Also, it is possible to elute resulted mRNA less than 100 ml, therefore increase mRNA concentration if it is necessary for other applications.
It is extremely important to complete mRNA isolation within 30 minutes of washing the magnetic beads. If you wait longer, the reaction yield will be smaller. It is also important to test the beads for uniformity before use, as advised in manual. However, all particles that I worked with settled in less than 3 minutes and still gave me good results. I would recommend that you pay strict attention to the particle's appearance. It should be uniform, no "dust" nor "clamps".
As conclusion, I would highly recommend to use this kit for mRNA isolation. It is convenient, produces mRNA free of other contaminant nucleic acid, its protocol is relatively quick and easy.