mRNA Isolation Directly from Lysates
The chemagic mRNA T Kit contains oligo(dT) molecules covalently bound to M-PVA Magnetic Beads enabling fast and convenient purification of mRNA from total RNA. Due to their high stability the magnetic beads are reusable and guarantee high yields of pure mRNA that can be used in e.g. RT-PCR amplification.
Left Fig.: Formaldehyde agarose gel. Electrophoresis of RNA from rat liver.
(1) 20 µg total RNA. (2) 10 µg isolated mRNA.
(3) 20 µg remaining RNA in supernatant following isolation.
(4) and (5) M-PVA OdT beads reused after regeneration. (4) 10 µg isolated mRNA. (5) 20 µg remaining RNA in supernatant.
Right Fig.: Northern blot with GAPDH probe.
Rows (1) - (5) correspond to left figure.
This rapid method for the purification of high quality mRNA has been optimized for direct purification of mRNA from crude extracts of animal tissues, cells, plants or blood. The direct isolation is performed without having to previously prepare total RNA or perform any other purification steps. The ease and speed of these kits has been optimized while maintaining the high yield and purities of the resultant mRNA. The use of the kit relies on the base pairing between the poly A tail of most messenger RNA and the oligo dT sequence bound to chemagen’s proprietary M-PVA Magnetic Bead surface. Comparison of the purity of mRNA isolated from a common sample using other available market kits with chemagens mRNA Direct Kit, showed that chemagen’s kit yielded the purest mRNA. The others show substantially more 18S and 28S rRNA contamination.
The use of chemagen M-PVA Magnetic Bead technology for direct mRNA isolation provides mRNA of high quality, yield, and purity in a very short time. The purified mRNA can be eluted off the beads in any convenient volume of elution buffer. The protocol is also readily adapted to microtiter plates for high throughput sample processing on robotic workstations. The chemagic mRNA Direct Kit guarantees a consistently high yield of excellent quality mRNA for downstream applications, such as RT-PCR.
