Berglund:Harvesting RNA

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HARVESTING RNA

1. Pre-warm 1X PBS in 37C for 20-30 minutes and put triple at room temperature.
2. While waiting for the 1X PBS to warm up, label 1.5mL eppendorf tubes and set them on ice for every well in which you will be harvesting.
3. In a 1.5mL eppendorf tube make RLT buffer. (Per 1mL of RLT add 10uL of BME). 350ul will be used per well.
4. Make cold 70% EtOH
5. Set up RNeasy spin columns.
6. Bring the 1.5mL eppendorf tubes that were put on ice to the tissue culture rooms (in the ice bucket).
7. In fume hood put 2 10mL pipette tips, set out the 50mL conical tube of OPTIMEM.
8. Take out 6 well plate and wash the wells 2 times with 2mLs of 1X PBS and aspirate off.
9. Add 450uL of tripleE per well and incubate for ~5 minutes at 37C.
10. Bang on the sides of the plate to help resuspend the cells.
11. Add 900uL of low growth media to each well. Transfer solution to the 1.5mL chilled eppendorf tubes.
12. Centrifuge for 45 seconds.
13. Aspirate off the supernatant.
14. Put cell pellets on ice and take back to the lab.

ISOLATING RNA FROM HeLa CELLS using the Qiagen RNAeasy Kit

1. To each pellet add 350uL of RLT + BME buffer, vortex and add 350uL of cold 70% EtOH. (10uL of BME/1mL of RT buffer)
2. Pipette to mix and transfer to an RNeasy kit spin column.
3. Centrifuge for 20 seconds at 10,000 rpm in counter top centrifuge by gel pouring station.
4. Discard flow-through into RNeasy waste and add 700uL of RW1 buffer.
5. Centrifuge for 20 seconds at 10,000 rpm.
6. Discard flow through in RNeasy waste and transfer spin-columns to new 2mL eppendorf tubes.
7. Add 500uL of RPE buffer to spin column and centrifuge for 20 seconds at 10,000 rpm.
8. Discard flow through into RNeasy liquid waste and add another 500uL of RPE buffer to the spin column and centrifuge for 2 minutes at 10,000 rpm.
9. Discard flow through into RNeasy liquid waste and put spin column in a new 2mL eppendorf tube and spin for 1 minute at 13,000 rpm.
10. Label 1.5mL eppendorf tubes (provided) and put spin columns in labeled 1.5mL eppendorf tubes.
11. Elute RNA by adding 30uL of RNase-free ddH2O from kit. Let sit for 1 minute and centrifuge at 10,000 rpm for 1 minute.
12. Put RNA in -80C

DNAse TREATMENT

1. Find concentrations of all RNA samples.
2. For each RNA sample a DNAse reaction will be done. Label 6 eppendorf tubes and add 1uL of 10X RQ1 DNAse buffer, 500ng of RNA, up to 9uL (total volume) of H20 and 1uL of RQ1 DNAse.
3. Incubate for two hours at 37C.
4. Keep on ice (or can store in -80C)