Real-time fluorescence quantitative PCR experiment operation process
1. Experimental equipment and reagents
1. Experimental equipment
Multi-sample grinding bead homogenizer
Desktop high-speed refrigerated microcentrifuge
Fluorescence quantitative PCR instrument
Clean bench
Spectrophotometer
Centrifuge tube
TIP header
2. Main experimental reagents and consumables
RNA extract
Trichloromethane
Isopropanol
Absolute ethanol
HyPure TMMolecular Biology Grade Water
RevertAid First Strand cDNA Synthesis Kit
FastStart Universal SYBR Green Master(Rox)
Primer
75% ethanol: HyPure TMMolecular Biology Grade Water
Centrifuge tube and TIP head are all purchased and sterilized by moist heat for 40 minutes, and then dried.
2. Fluorescence quantitative PCR experiment steps
1. Total RNA extraction (pipette tip and centrifuge tube are sterilized by moist heat, no RNase)
1) Take the homogenizer, add 1ml Trizol Reagent, and pre-cool on ice.
2) Take 100 mg of tissue and add it to the homogenizer.
3) Grind thoroughly until there is no visible tissue mass.
4) Centrifuge at 12000rpm for 10min to take the supernatant.
5) Add 250 μl of chloroform, invert the centrifuge tube for 15 seconds, mix well, and let stand for 3 minutes.
6) Centrifuge at 12000rpm for 10min at 4℃.
7) Transfer the supernatant to a new centrifuge tube, add 0.8 times the volume of isopropanol, and mix upside down.
8) Place at -20°C for 15 minutes.
9) Centrifuge at 12000 rpm for 10 min at 4°C. The white precipitate at the bottom of the tube is RNA.
10) Aspirate the liquid and add 1.5ml of 75% ethanol to wash the precipitate.
11) Centrifuge at 12000rpm for 5min at 4℃.
12) Absorb the liquid cleanly, place the centrifuge tube on the ultra-clean table and blow for 3 minutes.
13) Add 15μl of RNase-free water to dissolve RNA.
14) Incubate at 55°C for 5 minutes.
15) Use UV1800 to detect RNA concentration and purity: After the instrument is blanked and zeroed, take 2.5μl of the RNA solution to be tested on the detection base, put down the sample arm, and use the software on the computer to start the absorbance detection.
16) Dilute the RNA with an excessively high concentration in an appropriate proportion to a final concentration of about 200ng/μl.
2. Reverse transcription (both pipette tips and PCR are sterilized by moist heat, no RNase)
1) Take a PCR tube and add a solution containing 2μg RNA.
2) Add 1μl reverse transcription primer.
3) Make up to 12μl with deionized water without ribonuclease.
4) Incubate at 65°C for 5 minutes on the PCR machine, and quickly cool on ice.
5) Add 4μl of 5×buffer, 2μl of 10mM dNTPs, 1μl of RNA inhibitor and 1μl of reverse transcriptase in sequence, and mix well with a gun.
6) Incubate at 42°C for 60 minutes on a PCR machine, and then incubate at 80°C for 5 minutes to inactivate the reverse transcriptase.
3. Quantitative PCR
1) Take a 0.2ml PCR tube and prepare the following reaction system, with 3 tubes for each reverse transcription product.
2× qPCR Mix 12.5μl
7.5μM gene primer 2.0μl
2.5μl of reverse transcription product
ddH2O8.0μl
2) PCR amplification
Pre-denaturation 95℃, 10min
Cycle (40 times) 95°C, 15s→60°C, 60s
Melting curve 75℃→95℃, heating up 1℃ every 20s
4. Result processing
ΔΔCT method:
A=CT (target gene, sample to be tested)-CT (internal standard gene, sample to be tested)
B=CT (target gene, control sample)-CT (internal standard gene, control sample)
K=A-B
Expression multiple=2-K
