Real time qPCR (Fly Heads)
Mel Feany
Abstract
This Protocol describes how to perform RT qPCR with RNA extracted from fly heads.
Steps
RNA Extraction
Homogenize 6 heads in 50µL Qiazol, then add 450µL Qiazol.
Spin 0h 10m 0s at 4°C at 12,000 x g.
Incubate supernatant for 0h 5m 0s at Room temperature .
Add 100µL chloroform. Vortex, incubate for 0h 3m 0s at Room temperature , then spin for 0h 15m 0s at 4°C at 12,000 x g.
Transfer the upper phase to fresh tube, add 250µL isopropanol, incubate 0h 10m 0s at Room temperature , spin for 0h 10m 0s at 4°C at 12,000 x g.
Wash the pellet in 500µL 75% ethanol (it will be very loose) three times. Do not vortex.
Air dry
Resuspend in 15µL RNAse/DNAse-free water.
Measure 260/280 and 260/230 on nanodrop.
RT-PCR
Use 1µg RNA to perform RT-PCR reaction.
Thermocycler settings
a. 25°C 0h 10m 0s
b. 37°C 2h 0m 0s
c. 85°C 0h 5m 0s
d. 4°C Hold
qPCR
Make master mix with primers, SYBR Green and water.
Dilute DNA 1:50 making a 50µL stock.
Add 2µL DNA to wells with special tips.
Add 14µL master mix to wells with regular tips. Pipette up and down once but do not expel a bubble. Keep master mix on ice the entire time to avoid bubbles.
Check for bubbles and run on machine.
