The literature that comes with Stratagene’s Poly(A) Quik mRNA Isolation Kit states that these columns can accommodate up to 500ug of total RNA and yield purified mRNA for cDNA synthesis in about 15 minutes. The amount of mRNA that can be obtained ranges between 1 and 5 percent of the total RNA loaded. This kit is designed with push columns that eliminate slow flow rates and obstructed columns normally associated with gravity oligo(dT) chromatography.
Our lab deals in cDNA libraries, which require relatively pure mRNA as starting material. I have extracted mRNA from adherent cells in cell culture and various tumor tissues (colon, ovarian and breast tumors) as well as normal human and murine tissues (heart, lung, ovary, liver, stomach etc.). Yields of mRNA from cells grown in culture tend to be higher than mRNA yields from tumor or normal tissue (ranging from 1.5-2% of the total RNA loaded). My first attempts using the kit resulted in mRNA contaminated with ribosomal RNA. After consulting with Stratagene, the solution was to purify each sample twice. This procedure basically consists of pooling the elution aliquots and bringing them up to 1X with 10X Sample buffer. The sample is then reloaded on the same column and purified again. I found, however, that upon reloading the sample on the column the flow rate is greatly decreased. Superior results can be obtained using a new column for a second selection step instead of reloading the sample on the same column. After two selections, the ribosomal RNA disappears and high quality mRNA is obtained.
One of the features that I liked best about this kit is that it kept ribonuclease contamination to a minimum. Unlike other kits that I have worked with, the columns were easy to manipulate and keep clean. Other methods often involve free-floating slurries of oligo(dT) cellulose that are transferred from an ependorf to some sort of column to be spun in a centrifuge. More often than not, the columns were difficult to manipulate and handle effectively.
The Poly(A) Quik mRNA isolation kit is still time efficient, even with the second selection using a new column. Clean mRNA samples can be obtained in about 30 minutes. It is important to keep in mind that if the downstream application for the mRNA limits the sample volume, a precipitation step will be needed. Still, mRNA from tissue or cells can be obtained in one day. For my purposes, this was often helpful to advance the synthesis of a cDNA library. This kit is not cheap, but considering how precious the tissue samples we work with are, price isn’t a factor.
Alicia Racelis
Graduate Student
University of Alabama, Birmingham