mRNaid^® Kit Protocol
Oligo[dT]₃₀
Revision No. 2510-999-6C01W

In the isolation of mRNA, there are two methods that can be employed: The first is to isolate total RNA and then separate mRNA from it. This is more time consuming, but gives a better yield of poly(A+)mRNA (probably as high as five- to tenfold). The second method is to isolate the mRNA directly from a lysed supernatant without doing any prior extractions or separations. This method is faster and does not require any organic solvents for extractions, but it usually results in less yield. To its credit, however, this method is very much suited for applications not requiring a great yield, such as multiple samples for subsequent RT-PCR amplification.

During the entire procedure, except for the elution, the sample and solutions must be kept at 4°C (or on ice) and the magnetic beads must never be allowed to dry out. Act fast when pulling off the supernatants and resuspending the pellets.

Poly (A+) mRNA is approximately 2-5% of the total RNA yield from a given cell or tissue type. You will need to add 100 µl of the Matrix to 100 µg of total RNA for an expected yield of 2-5 µg of mRNA. These instructions are based on the isolation of poly (A+) mRNA from 100 µg of total RNA in a 300 µl maximum volume. The protocol can, however, be scaled up or down.

1. Wash 100 µl of Oligo-dT Matrix for each sample before use with 300 µl Bind Solution A. Pellet Matrix particles against the side of a microcentrifuge tube with the kit-supplied Single Tube Matrix Collector. Draw off supernatant. Resuspend Oligo-dT Matrix in 100 µl of Bind Solution A.



2. To an ice-cold RNA sample, add an equal volume of ice-cold Bind Solution B. Mix well. Add the washed Oligo-dT Matrix



3. Incubate on ice with occasional mixing for 10-30 minutes for hybridization of poly(A+)mRNA to the Oligo-dT Matrix. Longer times should be used for maximum yield.



4. Using the Matrix Collector, pull the Matrix/poly(A+)mRNA complex to one side of the tube and pipet off the supernatant that contains the unbound, total, non-polyadenylated RNA. Transfer the supernatant to another tube and save aside. Resuspend the pellet in 100 µl of fresh, ice-cold Bind Solution A and mix well while keeping the tube on ice during the entire procedure.



5. Wash the pellet three times with 300 µl/wash of ice cold Bind Solution A. (It is possible, at this point, to resuspend the pellet in the appropriate buffer and add the suspension directly to an RT-PCR reaction without prior elution of the poly (A+) mRNA from the Oligo-dT Matrix).



6. Elution: Elute mRNA by resuspending the pellet in 10-25 µl of hot, kit-supplied RNase-free Elution Solution (60-80°C). Incubate at 60-80°C for 2-5 minutes. Pull the Oligo-dT Matrix aside and remove the elution to a new tube. The elution now contains the polyadenylated RNA that is ready for enzymatic manipulations. Store at -70°C.

Again, keeping the solutions ice cold is essential for efficient binding.

1. Lyse the tissue or cells in a non-organic chaotropic reagent, e.g., 6 M guanidine thiocyanate. Add an equal volume of the Bind Solution B. Mix well.



2. Add 100 µl of the washed Oligo-dT Matrix for every 2 µg of mRNA yield (expected theoretical based on 2 to 5% poly (A+)mRNA to a given quantity of total RNA) to the lysate directly. Mix well and let incubate for 30-60 minutes on ice with occasional mixing.



3. Continue with step 4.