RNA Extraction with Trizol plus QIAGEN RNeasy mini kit
For cultured cells (3 to 5.106 cells).
Detailed protocol
- Pre-warm PBS to 37 °C and keep Trizol at room temperature.
- Aspirate medium, rinse the cell GENTLY with 1 ml of PBS.
- Aspirate PBS, add 1 ml of Trizol
- Scrape cells and transfer to 2 ml Eppendorf. Phase lock Gel Heavy, 2.0mL
- Add 200 µl of chloroform to each sample and mix gently.
- Centrifuge at 14000 rpm for 15 min at 4 °C.
- Recover the supernatant into a new Eppendorf.
- Add 350 µl of ethanol 100% to each sample, mix.
- Transfer all the solution into the column (QIAGEN RNeasy mini Kit): 700 µl at a time.
- Centrifuge at 14000 rpm for 30 s at room temperature, discard the flow-through.
- Add 350 µl of RW1 Buffer
- Centrifuge at 14000 rpm for 30 s at room temperature, discard the flow-through.
- For each sample, mix 10 µl of DNase (RNase-free DNase Set-QIAGEN) with 70 µl RDD Buffer, add 80 µl of this mixture to each sample (add to the center of the column).
- Wait for 15 min at room temperature.
- Add 350 µl of RW1 Buffer to the column.
- Centrifuge at 14000 rpm for 30 s, discard the flow-through.
- Add 500 µl of RPE Buffer (RPE Buffer is supplied as a concentrate. Ensure that 220 mL ethanols are added to RPE Buffer before use)
- Centrifuge at 14000 rpm for 30 s, discard the flow-through.
- Add 500 µl of RPE Buffer
- Centrifuge at 14000 rpm for 30 s, change to Collection Tube (2mL, without caps)
- Centrifuge at 14000 rpm for 1 min, change to Collection Tube (1.5 mL, with caps)
- Add 12-37.5 µl of RNase-free water, centrifuge at 14000 rpm for 30 s (don't through away the filtrate).
- Add 12-37.5 µl of RNase-free water, centrifuge at 14000 rpm for 1 min.
- Keep the RNA in freezer at 20 °C.