I first heard about Ambion in graduate school when a friend in the lab was purifying mRNA for a project he was working on. He had never done it before but he said that he had found a kit from a company that worked really well - he couldn’t remember the company’s name, but he said they called themselves “The RNA Company”. A few years later, I found myself looking for an mRNA isolation kit and I remembered his experience with “The RNA Company”, so I thought I’d give their Poly(A)Pure mRNA Isolation Kit a try. And I’m happy to say I wasn’t disappointed.
When you’re purifying mRNA there are usually 3 things on your mind (at least as far as mRNA isolation is concerned). They are (in no particular order) 1. quality (degraded mRNA is as close to useless as you can get) 2. purity (nothing worse than having to do multiple DNase digestions to get rid of that contaminating genomic DNA) and 3. yield. The Poly(A)Pure kit deals with these 3 points extremely well.
The Poly(A)Pure kit uses a standard GuSCN containing lysis solution to solubilize cells/tissue and inactivate endogenous RNases. The lysis solution is then diluted and the oligo dT cellulose is added. Ambion claims that their oligo dT cellulose not only gives the highest yield of mRNA but that it can bind to mRNA with as few as 8 adenylate residues in the polyA tail (ensuring that you’re not preferentially losing mRNA with short polyA tails). The oligo dT cellulose is then “batch-washed” sequentially with high-salt than low-salt wash solutions. The oligo dT cellulose is eventually collected in a spin-column and the mRNA is eluted with prewarmed elution buffer and is ready for any down-stream application.
The entire protocol for this kit is extremely easy, there’s nothing tricky about it, and the end result is a good yield of clean mRNA. The only down side is that there are a seemingly endless number of washes. The batch washes are performed in a 50ml conical tube and it invariably takes longer than you think it should. Still, it’s a small point and it’s not like you can cut the wash steps out. The only other issue that I have with this kit is that you only get enough oligo dT cellulose for 6 purifications (if you need to do more isolations, than have to order the oligo dT cellulose separately).
In the final analysis, I would have to say that Ambion’s Poly(A)Pure mRNA Isolation Kit is well worth using - I have consistently been able to obtain high yields of clean mRNA. It’s good to know that “The RNA Company” is living up to their name.
Michael Okimoto, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco