Prepare two ceramic beads tubes by filling a two milliliter PCR tube with an approximately 200 microliter tube full of 1.4 millimeter ceramic beads and label the tube accordingly before transferring them inside the sterile hood. Add the recommended volume of lysis buffer provided in the RNA extraction kit to the labeled PCR tube. Get approximately a three millimeter cube from the brain sample confirmed with rabies infection using a wooden applicator and put it into a labeled tube with sample ID and 100 microliters of nuclease-free water into the tube labeled negative control.
Disrupt the brain tissue manually using a wooden applicator stick and then vortex at maximum speed until complete tissue homogenization is achieved. Next, centrifuge the homogenized lysate. Transfer the supernatant to a newly labeled micro centrifuge tube using a pipette and use it for further RNA extraction steps, CDNA preparation, and PCR amplification.
After the CDNA preparation and PCR amplification using primer pools A and B, perform PCR cleanup and quantification in the post PCR area. Aliquot SPRI beads into micro centrifuge tubes from the main bottle. These can be also prepared in advance.
Store the tubes at four degrees celsius or keep them in a cold rack or ice. Next, warm the SPRI bead aliquot at approximately 20 degrees Celsius and thoroughly vortex to re-suspend the beads in the entire solution. Then in the 1.5 milliliter tubes, combine the primer pool A and primer pool B PCR products for each sample.
Add water to bring the volume to 25 microliters if required before adding 25 microliters of SPRI beads to each combined product and mixing it. Incubate the mixture at room temperature for 10 minutes with occasional inverting or flicking of the tubes. Next, place the tubes on a magnetic rack until the separation of beads and solution.
Once done, discard the supernatant without disturbing the bead pellet. Then give two washes of 30 seconds using freshly prepared 200 microliters of 80%ethanol and discard the ethanol. After the second wash, remove all traces of ethanol using a 10 microliter pipette tip.
Air dry the pellet until trace ethanol has evaporated and the pellet changes from shiny to matte. Re-suspend the beads in 15 microliters of nuclease-free water to recover the cleaned DNA product and incubate at room temperature for 10 minutes. Separate the beads from the solution on a magnetic rack.
After transferring the supernatant to a fresh 1.5 milliliter tube, prepare a one to 10 dilution of each sample in nuclease-free water. Measure the DNA concentration of each diluted sample with a highly sensitive and specific fluorimeter, following the manufacturer's instructions.
