The Dynal Direct mRNA Monocyte Isolation Kit utilizes Dynabeads technology to purify monocytes from whole blood and then isolate mRNA from those cells. Dynabeads are superparamagnetic, meaning they demonstrate magnetic properties when placed within a magnetic field (magnetic particle concentrator), but retain no residual magnetism when removed from the magnetic field. The kit includes two main dynabead preparations, antibody coated dynabeads against CD14 and Dynabeads Oligo (dT)
25.
Total monocytic RNA can be isolated from whole blood through the use of the CD14 coated dynabeads. Since CD14 is a well-characterized a monocytic marker, incubating whole blood with these beads allows monocytes to be isolated using the magnetic particle concentrator (not supplied). After the monocytes have been isolated, they are lysed and beads are removed. The sample is then incubated with the Oligo (dT)25 beads. These beads are 2.8um in diameter and have 25 nucleotide-long chains of deoxythymidines (dT) covalently linked to their surfaces via a 5’ linker group. This enables the purification of poly A+ mRNA from the total RNA.
All major reagents and buffers are supplied with the kit. The instructions supplied are relatively straight forward and easy to follow. A key feature that we found useful is that lysed monocytes can be frozen and stored and mRNA isolation, which takes around 15 minutes, can be performed at a later date. The one difficulty that we have found was that only 6 samples can be processed at any one time (with one magnetic particle concentrator), as the magnetic particle concentrator (MPC-E) can only hold 6 tubes and, if incubation times are to be regulated, more than 6 samples proves inefficient.
The mRNA isolated using the Dynabeads Oligo (dT)25 is in a concentrated form and may be used directly in all downstream applications. Within our lab we mainly used isolated monocytic mRNA for gene expression analysis via RT-PCR. This proved to be very successful with no contamination and without artifactual activation between samples as analysed by GAPDH expression.
The major advantages of this kit are obvious. It cuts down on many purification steps of both monocytes and total RNA therefore saving on time. Only a minute amount of starting RNA material is also needed as the hybridization step is extremely efficient. The isolation is convenient as only a single tube is needed, which reduces contamination problems. Enzymatic downstream applications are also not inhibited by the presence of the dynabeads. The major disadvantage of the kit is the price, which can prove expensive if a large number of samples are to be analysed and the initial outlay of the magnetic particle concentrator and equipment (rotor) if needed, increases the costs even further.
KJ Woollard, Ph.D.
Senior Research Officer
Baker Medical Research Institute
Australia