1.Wipe down lab bench and gloves with RNaseZap Solution wipes to avoid contamination. (provided with kit)
2. Pipette samples of Halo into 2 mL centrifuge tubes. (Since we had about 3 mL of Halo culture per sample, we split each falcon tube of Halo into two centrifuge tubes with about 1.5mL of sample each.) Centrifuge on high for 2 minutes.
3. Carefully pour of the supernatant so as not to dislodge the pellet.
4. Add 300 μL of Lysis/Binding solution to each tube, and vortex to completely mix. There should be no visible pieces of Halo cells.
5. Use a pipette to combine the tubes that hold the same sample into 1 2 mL tube, if you had to split your sample in step 2.
6. Add 1/10 of the volume of miRNA Homogenate Additive to the lysate and vortex for a few seconds or invert to mix. For example, since we had 600 μL from our combined tubes, we added 60 μL of additive.
7. Leave on ice for 10 minutes
8. Add a volume of Acid-Phenol:Chloroform in a fume hood that is equal to the original lysate volume before the miRNA Homogenate Additive. For example, since our volume before the Additive was 600 μL, we added 600 μL of Acid-Phenol:Chloroform. Be sure to draw from the bottom phase of the chloroform, because the upper phase is an aqueous buffer.
9. Vortex 30-60 seconds to mix
10. Centrifuge for 5 minutes at max speed (10,000 g) at room temperature
11. Carefully remove the aqueous (upper) phase with a 200 μL pipette in the fume hood and transfer to a fresh labeled tube. Use the pipette to figure out the largest volume removed from any of the samples.
12. Add 1.25 volumes of room temperature 100% ethanol to the aqueous phase. (This doesn’t have to be exactly 1.25. More is better, since it’s just washing the solution, so we added 1.25 of the greatest volume we measured to all of the tubes)
13. For each sample, place a filter cartridge in the collection tubes and pipette up to 700 μL of the lysate/ethanol mixture onto the filter. (The filter comes with the kit)
14. Centrifuge for 15 seconds at 10,000 g and discard the flow-through (liquid in the collection tube)
15. Repeat for the remaining ethanol/lysate mixture
16. Apply 700 μL miRNA wash solution 1 to the filter and centrifuge for 5-10 seconds. Discard the flow through.
17. Apply 500 μL wash solution ⅔ and centrifuge 5-10 seconds. Discard flow through.
18. Repeat step 17 with a 2nd 500 μL aliquot of wash solution ⅔
19. After discarding the flow through, put the filter back in the collection tube and spin down for one minute to remove residual fluid.
20. Transfer the filter to a fresh collection tube. Apply 100 μL of 95℃ heated elution solution or nuclease free water.
21. Spin for 20-30 seconds to remove the RNA from the filter, save the elute and store in -20℃.
22. Check RNA concentration and purity using nanodrop.
DNase treatment to further purify RNA samples:
1.Calculate how many μL of RNA would be needed to make 10 μg, based on the ng/μL concentration from the nanodrop numbers.
5 µL 10x buffer
5 μL DNase
X μL RNA (calculated in step 1)
X μL H2O (what’s needed to create 50 μL)
To make a 50 µL solution
3. Incubate samples in a water bath for 30 minutes at 37℃
4. Remove from the water bath, add 5 μL STOP solution
5. Incubate 10 minutes in a 60℃ water bath.
6. Add 1/10 of the volume of 3 M sodium acetate, 3 volumes of 100% ethanol, and 1 μL of glycogen blue. (The glycogen blue isn’t necessary, but makes the pellet more visible.)
7. Spin down and vortex to mix
8. Centrifuge at 4℃ and 15,000 rpm for 30 minutes
9. Pour off the supernatant, being careful not to dislodge the pellet. The pellet might be difficult to see, because the RNA is transparent (why the glycogen blue is helpful)
10. Add 150 μL of 70% ethanol to each tube
11. Centrifuge for 15 minutes at 4℃ and 15,000 rpm
12. Using a pipette with a 200 μL tip, remove the supernatant without disturbing the pellet. Use a 20 μL pipette to remove the last drop.
13. Dry for 10-15 minutes in a biosafety cabinet with the tube’s tops open. For faster drying, spin down quickly and use a 20 μL pipette to remove the extra ethanol.
14. Check the purity and concentration with the nanodrop and a bioanalyzer to confirm if your RNA is pure enough to use in RNAseq!
15. Store in -80℃.