Isolation of low amount of RNA with Trizol and glycogen
- perform the RNA isolation (trizol & choroform!) under fume hood and use gloves
- Pellet cells (in 1.5 or 2 ml tube) according to cell requirement (usually 5-10 min @ 4°C with 300 rcf)
- remove supernatant as much as possible (30 µl can be left)
- add 1 ml trizol (per 10^6 cells) and pipette mix thoroughly to proper lyse the cells
| optional: stopping point (4°C o/n, -20°C for up to 1 year) |
- incubate for 5 min at room temperature
- add 200 µl chloroform (under fume hood) and shake vigorously for 15 sec by hand
- incubate for 3 min at room temperature
- centrifuge samples 15 min @ 4°C with 14,000 rcf to get the phases separated1:
lower organic phase (red phenol-chloroform with proteins and lipids), interphase (DNA phase), aqueous upper phase (RNA) - transfer the aqueous upper phase into a new 1.5 ml tube, avoid transferring material from the interphase!
- optional for low cell numbers (<106): add 5-10 µg (1-2 µL) of RNase free glycogen
- precipitate RNA by adding 500 µL isopropanol
- mix thoroughly
- incubate for 10 min at room temperature
- centrifuge samples 10 min @ 4°C with 14,000 rcf
- remove supernatant as much as possible without disturbing the pellet (for low amount of RNA no pellet will be visible)
- add 1 mL 70 % ethanol to wash the RNA, vortex briefly
- centrifuge samples 10 min @ 4°C with 14,000 rcf
- remove supernatant completely and air-dry pellet for ~5 min
- dissolve in 5-8 µl nuclease free water (not DEPC treated)
Footnotes
pH of Trizol must be correct; DNA will remain in the upper phase when pH is too basic↩︎