Rapid mRNA Purification Kit

Directions for Use and Application Notes
Reagents Included in this Kit: Reagents and Equipment Needed (Not included with this kit):

  • Rapid mRNA Binding Buffer

  • Rapid mRNA Wash Buffer

  • Activated Oligo (dT) Cellulose

  • RNase-Free Water

  • Sterile, RNase-free microcentrifuge tubes

  • Micropipettor with RNase-free tips

  • Wide-bore yellow pipette tips

  • Microcentrifuge

  • 75C Water bath

  • Ice Bucket

  • 50-500g Purified Total RNA (A260/A280 > 1.7) in 100 l RNase-free water or 0.5% SDS

This kit is intended to isolate poly (A+) RNA from purified total RNA isolated from mammalian and plant cells and tissues. The procedure and volumes described will isolate up to 25 g poly (A+) RNA. This kit is not intended for purification of total RNA from cells or tissues. When using more than 500 g of purified total RNA as a starting material, volumes should be doubled. For best results, read the entire Directions for Use and all Application Notes before proceeding with this method.

 
Procedure  
  1. Before beginning this procedure the first time, add 500 l Rapid mRNA Binding Buffer to the lyophilized. Activated Oligo (dT) Cellulose. Vortex to mix. This slurry should be stored frozen when not in use.
  2. If total RNA is suspended in less than 100 L RNase-free water or 0.5% SDS,dilute sample to a total volume of 100 L with RNase-free water.

  3. Incubate RNA for 2 minutes at 75C.

  4. Re-suspend Oligo (dT) Cellulose slurry by brief vortexing.

  5. Add 100 l Rapid mRNA Binding Buffer and 20 l Oligo (dT) Cellulose slurry to the total RNA and vortex briefly to mix.

  6. Let tube stand on ice for 5 minutes, to allow for complete binding of the poly (A+) RNA to the Oligo (dT) Cellulose. Vortex the tube briefly once each minute.

  7. Microcentrifuge the tube for 10 seconds (pulse) to pellet the Oligo (dT) Cellulose with bound poly (A+) RNA. (See Application Note 3.)

  8. Remove and Discard the supernatant.Add 200 L of Rapid mRNA Wash buffer and vortex 5 seconds to mix.

  9. Microcentrifuge the tube for 10 seconds as before to pellet the solids.

  10.  Repeat Steps 8-9

  11. Add 20 L RNase-free water. Vortex briefly to mix, and incubate 30 seconds at 75°C.

  12. Microcentrifuge 10 seconds to pellet as before. Transfer supernatant to a new,sterile microcentrifuge tube.

  13. Add an additional 20 l RNase-free water to the pellet, vortex to mix, and repeat incubation and centrifugation steps.

  14. Transfer supernatant to the tube containing the supernatant from step 12.Store purified poly (A+) RNA at -80C.

 

Rapid mRNA Purification Kit -- Application Notes

Application Notes:

  • The Activated Oligo (dT) Cellulose is stable when stored lyophilized, frozen or at room temperature for up to six months. Once it has been reconstituted, it must be stored at -20C. Once thawed, the oligo (dT) cellulose may be difficult to pipette using a standard yellow tip. For best results, use a widebore pipette tip(similar to those used for genomic and lambda DNA preparation).

  • Complete denaturing of any secondary structure in the RNA is essential to efficient binding to the Oligo (dT) slurry. Ensure the water bath is pre-heated to 75°C, prior to beginning the purification protocol.

  • During centrifugation steps, it is important not to pellet the material for too long, as this may result in a pellet that is difficult to re-suspend. If your microcentrifuge has a pulse feature, a five to ten second pulse is sufficient to pellet the Oligo (dT) Cellulose.

  • It is recommended that the protocol be completed once begun. If, however, the protocol must be interrupted, be sure to place the tube containing the RNA on ice to help prevent degradation.

  • If a vortex mixer is not available, the pellets may be re-suspended by gentle pipetting using a sterile standard or wide bore pipette tips.

 

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