Fluorescence quantitative PCR experimental steps - Database & Sql Blog Articles

Fluorescence quantitative PCR experimental steps: 1. Sample RNA extraction

1. The frozen cells were thawed and allowed to stand at room temperature for 5 minutes to ensure complete dissolution.

2. Two-phase separation: Add 0.2 ml of TRIZOL reagent to each lysed sample, cap the tube, and shake manually for 15 seconds. Incubate at 15–30°C for 2–3 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. After centrifugation, the mixture separates into three layers: a red phenol phase, an intermediate layer, and a colorless aqueous phase. RNA is concentrated in the upper aqueous layer, which is about 60% of the volume of the TRIZOL reagent added.

3. RNA precipitation: Transfer the upper aqueous layer to a clean, RNase-free tube. Add an equal volume of isopropanol, mix for 10 minutes at 15–30°C, and centrifuge at 12,000 rpm for 10 minutes at 4°C. The RNA precipitates as a gelatinous pellet on the sides and bottom of the tube.

4. RNA washing: Remove the supernatant and add at least 1 ml of 75% ethanol (prepared in DEPC water) per 1 ml of the original TRIZOL solution. Mix well and centrifuge at 7,000 rpm for 5 minutes at 4°C.

5. RNA drying: Carefully remove most of the ethanol, and let the RNA pellet air-dry at room temperature for 5–10 minutes.

6. RNA resuspension: Dissolve the RNA pellet by adding 40 μl of RNase-free water and pipetting up and down several times. Store the RNA solution at -80°C until use.

Fluorescence quantitative PCR experimental steps, 2. RNA quality testing

1) UV absorption method

First, zero the spectrophotometer using a diluted TE buffer. Then, dilute the RNA solution with TE (1:100), measure the absorbance at 260 nm and 280 nm to determine concentration and purity.

1. Concentration determination

A reading of 1 at A260 corresponds to 40 μg RNA/ml. The RNA concentration is calculated as: A260 × dilution factor × 40 μg/ml.

Example: RNA dissolved in 40 μl DEPC water, 5 μl taken and diluted 1:100 to 495 μl TE. Measured A260 = 0.21

RNA concentration = 0.21 × 100 × 40 μg/ml = 840 μg/ml or 0.84 μg/μl

After measuring 5 μl, 35 μl of RNA remains. Total RNA amount = 35 μl × 0.84 μg/μl = 29.4 μg

2. Purity detection

The ratio of A260/A280 indicates RNA purity. A good range is between 1.8 and 2.1.

2) Denaturing agarose gel electrophoresis

1. Gel preparation: Dissolve 1 g agarose in 72 ml water, cool to 60°C, then add 10 ml of 10× MOPS buffer and 18 ml of 37% formaldehyde (12.3 M). Pour into a gel tray and leave at least 25 μl for the sample well. After solidification, remove the comb and place the gel in an electrophoresis tank with enough 1× MOPS buffer to cover the gel.

2. RNA sample preparation: Take 3 μg of RNA, add 3 times the volume of formaldehyde loading dye, and add ethidium bromide (EB) to a final concentration of 10 μg/ml. Heat at 70°C for 15 minutes to denature the RNA.

3. Electrophoresis: Pre-run the gel for 5 minutes before loading the samples. Run at 5–6 V/cm for 2 hours, until the bromophenol blue marker has migrated at least 2–3 cm.

4. Observation and imaging: Under UV light, the 28S and 18S ribosomal RNA bands should be clear and distinct. The 28S band is usually twice as intense as the 18S. A faint band of low molecular weight RNA (like tRNA and 5S rRNA) may also be visible. If DNA contamination occurs, it will appear as a higher molecular weight smear. Degraded RNA shows a smeared pattern. Capture the image with a digital camera.

3. cDNA synthesis

Fluorescence quantitative PCR experimental procedure

1. Reaction system

Reagents:

1. Reverse transcription buffer – 2 μl

2. Forward primer – 0.2 μl

3. Reverse primer – 0.2 μl

4. dNTPs – 0.1 μl

5. Reverse transcriptase (MMLV) – 0.5 μl

6. DEPC water – 5 μl

7. RNA template – 2 μl

8. Total volume – 10 μl

Mix the reaction components thoroughly, briefly centrifuge at 6000 rpm, and proceed to the next step.

2. Dry the mixture in a dry bath at 70°C for 3 minutes before adding the reverse transcriptase. Immediately after removal, cool the tube in an ice water bath. Add 0.5 μl of MMLV and incubate at 37°C for 60 minutes.

3. After incubation, heat the reaction at 95°C for 3 minutes to terminate the reaction. This produces a cDNA solution that can be stored at -80°C for later use.