To begin, prepare 10 to 20 micrograms of high-quality total RNA for the isolation of poly(A)enriched RNA. Transfer an aliquot of RNA into a nuclease free 1.5 milliliter microcentrifuge tube. Incubate the tube in a heat block at 70 degrees Celsius for five minutes, and then place it on ice for two minutes.
After resuspending the oligo(dT)beads, transfer the required volume of bead suspension to a new 1.5 milliliter microcentrifuge tube and place it on the magnet for three minutes until the magnetic beads form a pellet. Remove the supernatant and add an equal volume of lysis buffer. Resuspend the beads.
Place the tube back on the magnet for three minutes and remove the supernatant. For the first round of purification, add lysis buffer to the RNA sample tube and mix thoroughly. Then transfer the mixture to the oligo(dT)beads and resuspend fully pipetting at least 10 times.
Allow the samples to incubate with continuous rotation at room temperature. After 15 minutes, place the sample on a magnet for five minutes or until the supernatant is clear. Discard the supernatant.
Add 600 microliters of wash buffer one to the beads and carefully mix to resuspend. Next, add 300 microliters of wash buffer two to the beads and carefully mix to resuspend. Place the tube on the magnet for five minutes or until the supernatant is clear.
Discard the supernatant. Next, add 30 microliters of cold 10 millimolar tris HCL to the sample tube and incubate at 70 degrees Celsius. After five minutes, quickly transfer the tube on the magnet.
And when the suspension is clear, transfer the supernatant containing the eluted poly(A)enriched RNA into a new 1.5 milliliter microcentrifuge tube. For the second round of purification, add 120 microliters of lysis buffer to the eluted RNA sample and mix thoroughly. Wash the beads used in the first round of purification with an equal volume of lysis buffer.
Pipette 10 times to resuspend the beads and then place the tube on the magnet for five minutes before discarding the supernatant. Transfer the RNA lysis buffer mixture to the washed beads and mix thoroughly by pipetting. After washing the beads as demonstrated previously, add 25 microliters of cold 10 millimolar tris HCL to the sample tube and incubate at 70 degrees Celsius.
After five minutes, quickly transfer the tube to the magnet, and when the suspension is clear, transfer the supernatant containing eluted poly(a)enriched RNA to a new 1.5 milliliter microcentrifuge tube. Next, for bisulfite conversion, transfer 19 microliters of purified poly(A)enriched RNA sample to a 0.2 milliliter eight strip tube and add one microliter of spiking control at the predetermined one to 10, 000 quantity ratio. After adding 130 microliters of conversion reagent to the tubes and thoroughly mixing, place the tube in the PCR machine and start the PCR program.
After PCR, place the column onto the collection tube and add 250 microliters of RNA binding buffer to the column. Then transfer 150 microliters of bisulfite treated sample to the column and pipette thoroughly. Add 400 microliters of 95 to 100%ethanol to the column, quickly close the cap and invert several times.
After repeated centrifugation, washing, and neutralization with desulphonation buffer, transfer the column to a new 1.5 milliliter microcentrifuge tube. Add 20 to 30 microliters of nuclease-free water to the column and allow it to stand for one minute. Centrifuge at 10, 000 G for 30 seconds at 25 degrees Celsius and transfer 2.2 microliters of supernatant to eight strip tubes to assess RNA quantity and quality.
Poly(A)RNA enrichment efficiency was assessed by capillary electrophoresis total RNA assay, resulting in decreased ribosomal RNA contamination after double purification in pancreatic cancer cell lines. RNA quantity before and after bisulfite treatment was assessed by capillary electrophoresis. After the bisulfite treatment, the RNA size distribution showed a 200 to 500 nucleotide peak because of fragmentation caused by the bisulfite reaction.
Capillary electrophoresis of the amplicon showed successful library preparation with minimal primer remaining and no over amplification peak. After the sequencing reads aligned to the reference sequence, the average of 2, 440 of total reads were mapped to the spiking sequence, and the total analyzed C to T conversion rate reached an average of 99.81%
