C. elegans RNA Isolation and RT-PCR
Reagents Needed:
M9 (common stock)
Trizol (stored at 4ºC)
chloroform
2-propanol
70% EtOH
RNase-free H2O
iScript cDNA Synthesis Kit
Ambion DNA-free Kit
BioRad iQ SYBR Green Supermix
Procedure:
* Wear gloves at all times.
* Only use filter tips.
* Keep everything on ice as much as possible to avoid RNA degradation.
I. RNA Isolation
1. Pick 10 worms into 20μL of M9 in an RNase-free Eppendorf tube.
2. Pellet worms by spinning briefly at 14,000 rpm.
3. In the hood, add 250μL of Trizol.
4. Vortex by hand for about 30 seconds and then vortex at 4ºC until the worms
dissolve (about 20 minutes)
Optional (for large samples):
a. Let sit at room temperature for 10 minutes.
* At this point the worms can be frozen. Remove excess supernatant and
flash freeze in liquid nitrogen or dry ice/EtOH. Store samples at -70ºC.
b. Centrifuge at 14,000 rpm for 10 minutes at 4ºC.
c. Transfer the supernatant into an RNase-free Eppendorf tube.
5. In the hood, add 50μL of chloroform.
6. Vortex for 30 seconds.
7. Let sit at room temperature for 3 minutes.
8. Centrifuge at 12,000 rpm for 15 minutes at 4ºC.
9. Transfer the clear top layer (~125μL) into a new RNase-free Eppendorf tube.
10. Repeat steps 5-9.
11. Add 125μL of 2-propanol and invert to mix.
12. Let sit at room temperature for several minutes.
13. Spin down at 12,000 rpm for 10 minutes at 4ºC.
14. Carefully decant the supernatant. Leave a few μLs at the bottom so as not to
disturb the pellet.
15. Wash pellet with 250-500μL of 70% EtOH (use RNase-free H2O).
16. Spin down at 14,000rpm for 5 minutes at 4ºC.
17. Remove as much supernatant as possible and air dry pellet or use the air vacuum.
18. Dissolve the pellet in 10μL of RNase-free H2O.
19. Optional: Heat for 10 minutes at 60ºC to help dissolve the RNA.
Optional: RNA/DNA Contamination Quantification
* You will not get an accurate reading with small samples.
OD260/OD280: 1.8 for pure DNA
2.0 for pure RNA
Sample should be somewhere in between.
Make a 1:1,000 dilution
[RNA] = (OD260)x(40 μg/mL)x1000
[Total RNA] = [RNA]x19
II. DNA-Free Protocol (Ambion Kit)
1. For a 20μL reaction, add to an RNase-free Eppendorf tube:
10μL RNA
2μL 10x DNase I buffer
7μL RNase-free H2O
1μL DNase I
2. Incubate at 37ºC. for 20-30 minutes.
3. Add 2.5μL of DNase Inactivation Reagent.
4. Incubate for 2 minutes at room temperature with occasional mixing.
5. Spin down at 14,000 rpm for 1.5 minutes at 4ºC.
6. Transfer supernatant, without disturbing the white pellet, to a new RNase-free
Eppendorf tube.
Optional: RNA Quantification
* You will not get an accurate reading with small samples.
Make a 1:1,000 dilution
[RNA] = (OD260)x(40 μg/mL)x1000
[Total RNA] = [RNA]x49
III. cDNA Synthesis
1. For one 20μL sample, add:
4μL 5x iScript Mix
1μL Reverse Transcriptase
10μL RNA
5μL RNase-free H2O
2. Program:
25ºC for 5 minutes
42ºC for 30 minutes
85ºC for 5 minutes
4ºC ∞
3. Store at 4ºC or -20ºC until needed.
IV. RT-PCR
1. For one 25μL sample, add to one well of a 96-well plate:
1.5μL cDNA
0.5μL Forward primer (1:10)
0.5μL Reverse primer (1:10)
12.5μL CybrGreen buffer
10.0μL sterile dH2O
2. Pipet each sample in triplicate.
3. Use the Biorad iCycler in the Keck Biophysics Facility on the 4th floor of Cook.
NOTE: You must sign up to use the iCycler and either be trained to use the
machine or go with someone who knows how.
Recipes:
M9 (1L)
* Common lab stock in worm room.
5.8g Na2HPO4•7H2O
3.0g KH2PO4
5.0g NaCl
0.25g MgSO4•7H2O
ddH2O to 1L
• Filter (0.22μm) and bottle.
Reference:
This protocol has been modified by many Morimoto lab members over the years.
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