In this step, RNA from dried whole blood collected in Mitra tips is extracted by incubating with RNA Extraction Master Mix, purified by provided magnetic beads, and eluted from the beads by Hybridization Master Mix. For the RNA extraction step, we recommend using 1.5 ml test tubes. Please note that the proposed volumes of Master Mixes for all follow-up steps in the protocol already include 10-20% excess of reagents, therefore it is not necessary to increase the volume of reagents to prepare Master Mixes.
- Prepare RNA Extraction Master Mix as described below for all samples and aliquot 230 µl in each 1.5-ml test tube.
RNA Extraction Master Mix Component Volume per sample, µl Extraction Buffer, 1X 240 Proteinase K, 25x 10 Total 250
- Insert Mitra tip in 1.5 ml test tube with aliquoted RNA Extraction Master Mix and release from the holder by pushing the tip down using a 200-µl pipet tip. As a result, the released Mitra tip will be covered by the Extraction Master Mix.
- Close the test tube, insert it in the test tube shaker, and shake the tube at 1200 rpm at 60°C for 15 min, followed by 80°C for an additional 15 min.
- Using a 200-µl pipette, remove 200 µl of the RNA Extraction Master Mix (now containing extracted RNA) and transfer it to a new 1.5 ml test tube. Discard the test tube with the Mitra tip.
- Prepare Magnetic Bead Master Mix as described below for all samples:
Magnetic Bead Master Mix Component Volume per sample, µl Isopropanol (100%) 125 Magnetic Beads 2 Total 127
- Aliquot 120 µl Magnetic Bead Master Mix in 1.5 ml test tube with extracted RNA. Incubate the test tube in the shaker at 1200 rpm at room temperature for 5 min.
- Place the test tubes in the Magnetic Stand for 1-2 minutes or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet.
- Add 800 μl of MB Wash Buffer in each test tube without removing the test tubes from the Magnetic Stand, wait for 2 minutes, carefully remove, and discard the supernatant without disturbing the bead pellets.
- Repeat the 800 μl washing step with MB Wash Buffer as in the above Step.
- Repeat the 800 μl washing step with freshly prepared 80% ethanol as in the above Step.
- Briefly centrifuge the test tubes at low speed and place the plate in the Magnetic Stand. Use a 20-μl pipette to remove the residual ethanol droplets from tubes 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 magnetic beads.
- Prepare a Hybridization Master Mix as described below for all samples and one positive control RNA sample.
Hybridization Master Mix Component Volume per sample, µl Hybridization Buffer, 4X 6 Reverse hGW19K primer mix, 5X* 4.8 Water 13.2 Total 24
- Add 22 μl of the Hybridization Master Mix in each test tube and resuspend magnetic beads by gentle pipetting without generating bubbles. Briefly centrifuge the tubes at low speed and place the plate in a Magnetic Stand for 2 minutes. Transfer 20 μl of clear supernatant without beads from each test tube in individual wells on a 96-well plate or in the 0.2-0.5-ml test tubes (for a small number of samples) for the follow-up step. Seal the plate with adhesive film and spin it down to collect droplets.
- Human PBMC RNA is supplied with the kit as a positive control. We recommend running one positive control sample with your experimental samples. This will help with troubleshooting, data analysis, and normalization across different sample runs. You could also consider running the Hybridization Master Mix (without adding RNA) as a negative control.
- For positive control, aliquot 20 µl of Hybridization Master Mix in a well/test tube and add 1 µl (50 ng) of Positive Control PBMC RNA.
Last modified:
23 December 2024
Need more help with this?
Contact Us