The PolyAttract® mRNA isolation kits from Promega are an affordable and easy-to-use system for isolating mRNA using a magnetic approach. Products are available for small and large scale isolations and can be purchased with or without the magnetic stand. The system uses a neodymium cobalt magnet to attract the streptavidin paramagnetic particles. A biotinlyated oligo-dT probe hybridizes to the mRNA, which is subsequently attracted by the magnetic particles. The high affinity interaction between biotin and streptavidin allows the system to isolate relatively pure mRNA. The procedure essentially involves allowing the mRNA with the biotinylated oligo-dT to bind to the particles, washing the particles, and eluting the mRNA in RNase free water.
The whole procedure takes less than hour and does not require extensive optimization. Another advantage is that the particles have a relatively long shelf-life, reducing the likelihood of the reagents going bad in the refrigerator. The method does not require a column so there is no problem with clogging or painfully slow flow through the column. The particles come in individual aliquots so there is nothing to measure. The small scale kit is designed for isolation of mRNA from 100 ìg to 1 mg of total RNA and the large scale kit from 1-5 mg of total RNA. The larger scale is ideal for people who need a lot of mRNA from a single sample, whereas the smaller scale is perfect for people who need smaller amounts of mRNA from several samples.
I used the mRNA that I prepared with this kit for reverse transcriptase(RT)-PCR with amplicons as large as 4 kb and saw bright bands on the gel. Therefore, I would imagine that the mRNA would be suitable for most RT-PCR applications. Because I was able to amplify such large fragments without difficulty, the mRNA should be acceptable for applications such as Northern blots where intact RNA is critical. I started with approximately 100 ìg of total RNA and ended with a final yield of about 3 ìg of mRNA, which agrees with the theoretical percentage of mRNA in total RNA. The best part of the procedure is that it worked on the first try. Because polyA RNA isolation requires so much precious RNA, it is imperative that the procedure work without much tweaking.
The magnetic stand only holds 2 tubes so it might seem inconvenient to process several samples at one time. However, it takes less than a minute for the magnetic particles to separate so more than 2 samples can easily be processed together. Because the particles need to be used within 30 minutes after they are washed, it is not possible to reuse them as it is with oligo-dT cellulose. Fortunately, the kits are reasonably priced so this drawback is relatively minor. Likewise, the convenience of having the particles in individual aliquots limits the ability to scale the reactions, although there is quite a bit of flexibility in how much RNA can be used with a single aliquot.