RT-PCR using Trizol and Superscript III
Use RNAse free plastics and solutions!
Worm collection and homogenization
Collect 20 - 100 worms in an eppendorf tube with PBST. The number of worms will depend on the number of PCR reactions you will perform. More worms per PCR will lead to a better estimation of the average amount of DNA. Spin the worms down, and remove as much supernatant as possible using first a regular pipet and then a mouth pipet fitter with a draw out capillary. Freeze and thaw the worms twice in liquid nitrogen. Add 500 µl Trizol (Invitrogen #15596-026), and incubate for 15 minutes at room temperature to dissociate nucleoprotein complexes. Here you can store the mixture frozen at -80°C for up to one month.
Add 100 µl Chloroform, and shake the tube vigorously by hand for 15 seconds. Incubate for 2 minutes at room temperature, then spin at 12,000 g for 15 minutes at 4°C. After centrifugation, the lower red, phenol-chloroform phase contains protein, the interphase contains DNA and the aqueous upper phase contains RNA. Transfer the upper phase to a new tube. Optionally you can add 5-10 µg RNAse-free glycogen (Invitrogen #10814) as carrier. Add 250 µl Isopropanol, mix and incubate at room temperature for 10 minutes. Spin at 12,000 g for 15 minutes at 4°C. Unfortunately you will likely not see a pellet. Remove the supernatant, and wash the pellet with 75% Ethanol. To resuspend the RNA, dry the tubes briefly and add 50-100 µl DEPC treated water. Heat for 10 minutes at 55°C.
These are the instructions for the SuperScript III with Platinum Taq kit (Invitrogen #12574-026). All assembly should be performed on ice. The PCR machines should be allowed to heat to 50°C first before adding the tubes. Assemble the following reaction:
|2x Reaction mix||12.5 µl|
|Template RNA||x µl|
|Sense primer (10 pmol/µl stock)||0.5 µl|
|Antis-ense primer (10 pmol/µl stock)||0.5 µl|
|Superscript III enzyme mix||1 µl|
|Water||to 25 µl|
Run the following program on the PCR machine. The number of amplification cycles varies. A good set to try is 30, 35 and 40 cycles.
- 1 cycle:
- 50°C for 30 min.
- 1 cycle:
- 94°C for 2 min.
- 30-40 cycles:
- 94°C for 15 sec.
- 55-65°C for 30 sec.
- 68°C for 1 min/kb.
- 1 cycle:
- 68°C for 5 min.