線蟲總RNA提取及RT-PCR.doc

C. elegans RNA Isolation and RT-PCRReagents Needed:M9 (common stock)Trizol (stored at 4C)chloroform2-propanol70% EtOHRNase-free H2OiScript cDNA Synthesis KitAmbion DNA-free KitBioRad iQ SYBR Green SupermixProcedure:* Wear gloves at all times.* Only use filter tips.* Keep everything on ice as much as possible to avoid RNA degradation.I. RNA Isolation1. Pick 10 worms into 20L of M9 in an RNase-free Eppendorf tube.2. Pellet worms by spinning briefly at 14,000 rpm.3. In the hood, add 250L of Trizol.4. Vortex by hand for about 30 seconds and then vortex at 4C until the wormsdissolve (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 andflash freeze in liquid nitrogen or dry ice/EtOH. Store samples at -70C.b. Centrifuge at 14,000 rpm for 10 minutes at 4C.c. Transfer the supernatant into an RNase-free Eppendorf tube.5. In the hood, add 50L 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 4C.9. Transfer the clear top layer (125L) into a new RNase-free Eppendorf tube.10. Repeat steps 5-9.11. Add 125L 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 4C.14. Carefully decant the supernatant. Leave a few Ls at the bottom so as not todisturb the pellet.15. Wash pellet with 250-500L of 70% EtOH (use RNase-free H2O).16. Spin down at 14,000rpm for 5 minutes at 4C.17. Remove as much supernatant as possible and air dry pellet or use the air vacuum.18. Dissolve the pellet in 10L of RNase-free H2O.19. Optional: Heat for 10 minutes at 60C 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 DNA2.0 for pure RNASample should be somewhere in between.Make a 1:1,000 dilutionRNA = (OD260)x(40 g/mL)x1000Total RNA = RNAx19II. DNA-Free Protocol (Ambion Kit)1. For a 20L reaction, add to an RNase-free Eppendorf tube:10L RNA2L 10x DNase I buffer7L RNase-free H2O1L DNase I2. Incubate at 37C. for 20-30 minutes.3. Add 2.5L 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 4C.6. Transfer supernatant, without disturbing the white pellet, to a new RNase-freeEppendorf tube.Optional: RNA Quantification* You will not get an accurate reading with small samples.Make a 1:1,000 dilutionRNA = (OD260)x(40 g/mL)x1000Total RNA = RNAx49III. cDNA Synthesis1. For one 20L sample, add:4L 5x iScript Mix1L Reverse Transcriptase10L RNA5L RNase-free H2O2. Program: 25C for 5 minutes 42C for 30 minutes 85C for 5 minutes 4C 3. Store at 4C or -20C until needed.IV. RT-PCR1. For one 25L sample, add to one well of a 96-well plate:1.5L cDNA0.5L Forward primer (1:10)0.5L Reverse primer (1:10)12.5L CybrGreen buffer10.0L sterile dH2O2. 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 themachine or go with someone who knows how.Recipes:M9 (1L)* Common lab stock in worm room.5.8