Quantitative Real-Time PCR

Materials and Reagents

1. Human glioma tissues
2. Chloroform
3. Isopropyl alcohol
4. 75% DEPC-ETOH
5. RNase-free H₂O
6. HIScript^® II Q RT SuperMix (#R233-01, Vazyme)
7. Trizol (Life Technologies, Invitrogen™)
8. ChamQ SYBR Master Mix (#Q311-02/03, Vazyme)
9. Oscillator
10. 10.Nanodrop

Equipment

1.Biosystems StepOnePlus^TM Real-Time PCR System

Procedure

1.1 Total RNA extraction (for glioma tissues)

1.1.1. Glioma specimens were removed from liquid nitrogen, and patient information was recorded subsequently. Add 1 ml of Trizol to lyse glioma tissues by grinding. Incubate at room temperature for 20 min.

1.1.2. Add 0.2 ml of chloroform and cap tubes securely. Shake tubes vigorously by oscillator for 15 sec and incubate at room temperature for 2 to 3 min. Centrifuge at 12,000 g for 15 min at 4 °C.

1.1.3. Carefully transfer the top aqueous phase (about 0.6 ml) to a new clean 1.5ml tube.

1.1.4. Add 0.6 ml chloroform to the aqueous phase and cap tubes securely. Shake tubes vigorously and incubate at room temperature for 2 min and centrifuge at 12,000 g for 15 min at 4 °C.

1.1.5. Carefully transfer the top aqueous phase to a new clean tube.

1.1.6. Precipitate RNA by adding equal volume of isopropyl alcohol to the aqueous phase and mix well. Incubate at room temperature for 10 min. Centrifuge at 12,000 g for 10 min at 4 °C.

1.1.7. Remove supernatant and wash RNA with 1 ml of 75% DEPC-ETOH. Mix the sample by vortexing, and centrifuge at 7,500 g for 5 min at 4 °C.

1.1.8. Remove the supernatant and briefly air dry the RNA pellet ( no more than 10 min). 

1.1.9. Dissolve RNA in RNase-free H2O by pipetting. Incubate at room temperature for about 5-10 min. 

1.1.10. Measure RNA concentration using Nanodrop. 

1.1.11. Check RNA quality by A260/A280 and A260/A230 (RNA with good quality is close to 2 for both). Store RNA at -80 °C.

1.2 Remove genomic DNA (HIScript^® II Q RT SuperMix).

High-quality intact RNA is essential to obtain high-quality cDNA. The integrity of the RNA needs to be verified before the experiment.

Prepare the following mixture in an RNase-free centrifuge tube:

                                                          *Gently mix with a pipette. 42°C for 2 min.

1.3 cDNA synthesis (HIScript^® II Q RT SuperMix)

1.3.1. Add 5×qRT SuperMix II directly to the reaction tube from step 1.2

* Gently mix with a pipette.

1.3.2. Perform reverse transcription reaction

1.4 qPCR reaction (ChamQ SYBR Master Mix)

1.4.1. Prepare the following mixture in a qPCR tube

* Gently mix with a pipette. Total volume is 10 µl.

1.4.2. Perform qPCR reactions under the following conditions

1.5 Collect data and analyze the results.

β-actin gene was used as a control to normalize the results. In order to facilitate the subsequent difference comparison, we use the following formula:

Target gene relative expression = number of cyclic reaction repetitions / (target gene cycle number - β-actin gene cycle number)