6.1. Hybridization of mRNA with Reverse Gene-Specific Primers

In this step, Reverse hGW19K primers (Rev GSPs) are hybridized with target mRNAs, and mRNA-Rev GSP hybrids are purified from non-hybridized primers by SPRIselect magnetic beads. Both purified RNA and purified cells (Section 5.2 DirectCell protocol) can be used in this protocol. The protocol assumes that the reactions will be set in a 96-well PCR plate. For small numbers of samples, reactions can be done in tubes or strips.

*Note: Hybridization, cDNA synthesis, and Forward Primer Extension steps should be set up in a pre-PCR room to avoid contamination with final PCR products.

1. Prepare a Hybridization Master Mix as described below for all samples and controls (make 5% extra to account for pipetting error) and aliquot 9 µl in the wells of a 96-well plate:
    
   

   Hybridization Master Mix Component	Volume per sample, µl
   Hybridization Buffer, 4X	5
   Reverse hGW19K primer mix, 5X*	4
   Total	9

*Note: If you decide to run biological replicates, increase the number of tubes, volume, and amount of RNA allocated for each sample respectively.

2. Adjust the volume of each RNA Sample (e.g., 50 ng of RNA) to 11 µl with water as shown in the table or directly use 11 µl of RNA with low concentration (e.g., 5 ng/ µl).
    
   

   Component	Volume per sample, µl
   Total RNA (~50 ng)	1 – 11*
   Water, to 11 µl final volume	0 – 10

*Note: As a positive control, we recommend running 50 ng of positive control RNA included in the kit; and as a negative control, 11 µl of water.

3. Add 11 µl of RNA Sample to each well with the pre-aliquoted 9 µl of Hybridization Master Mix and mix contents using an Eppendorf plate shaker or by pipetting 3 times. Seal the plate with adhesive film and spin it down to collect droplets. Load the plate in the thermal cycler, and run the following program to hybridize mRNAs with Reverse hGW19K primers:
    
   

   Temperature	Time
   70°C	5 min
   60°C	60 min
   25°C	∞

4. Add 24 µl (1.2x volume) of SPRIselect magnetic beads (adjusted to room temperature) to each reaction well, seal the plate, and mix SPRIselect beads and hybridization solution in wells using Eppendorf plate shaker. Importantly, you need to optimize the speed of the plate shaker (e.g., 16,500 rpm for 30-60 sec) to completely mix viscous SPRIselect beads with solution in each well. Check visually that each well contains a homogeneous brown color solution in the whole volume.

5. Incubate the mixture for 5-10 minutes at room temperature. While waiting, prepare the Reverse Transcriptase Master Mix based on the protocol in Step 6.2 and store on ice before use.

6. Place the plate in the Magnetic Stand for 96-well plates for 1-2 minutes or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet.

7. Add 200 μl of freshly prepared 80% ethanol in each reaction well without removal of the plate from the Magnetic Stand, wait for 2 minutes, carefully remove, and discard the supernatant without disturbing the bead pellets.

8. Repeat 80% ethanol washing as in Step 7.

9. Briefly centrifuge the plate at low speed and place the plate in the Magnetic Stand. Use a 20-μl pipette to remove the residual ethanol droplets from reaction wells and air-dry the beads at room temperature for approximately 5 minutes. Visually check that no residual ethanol droplets are present in the wells. Avoid over-drying the SPRI beads.

!Immediately proceed to the next step (cDNA Synthesis)