Fluorescence quantitative PCR experimental steps - Database & Sql Blog Articles

Fluorescence quantitative PCR experimental steps: 1. Sample RNA extraction

1. The frozen cells were thawed 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 lower red phenol phase, an intermediate layer, and an upper colorless aqueous phase where RNA is concentrated. The upper aqueous phase typically constitutes about 60% of the volume of the TRIZOL added during homogenization.

3. RNA precipitation: Transfer the upper aqueous layer to a clean, RNase-free centrifuge tube. Add an equal volume of isopropanol and mix for 10 minutes at 15–30°C. Centrifuge at 12,000 rpm for 10 minutes at 4°C. At this point, the RNA will precipitate as a gel-like substance on the bottom and sides 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 lysed sample. Mix well and centrifuge at 7,000 rpm for 5 minutes at 4°C.

5. RNA drying: Carefully remove most of the ethanol and allow the RNA pellet to 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 resulting RNA solution at -80°C until use.

Fluorescence quantitative PCR experimental steps: 2. RNA quality testing

1) UV absorption method:

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

1. Concentration determination:

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

Example: RNA was dissolved in 40 μl of DEPC water. Take 5 μl, dilute it 1:100 with TE to a total of 495 μl. A260 = 0.21.

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

The remaining RNA volume after measurement is 35 μl, so the total RNA amount is 35 × 0.84 = 29.4 μg.

2. Purity detection:

The ratio of A260/A280 indicates RNA purity. A good ratio ranges from 1.8 to 2.1.

2) Denaturing agarose gel electrophoresis:

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

2. RNA sample preparation: Take 3 μg of RNA, add 3× volume of formaldehyde loading dye, and stain with 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 the gel at 5–6 V/cm for 2 hours, until the bromophenol blue indicator has migrated at least 2–3 cm.

4. Observation and imaging: Under UV light, the 28S and 18S ribosomal RNA bands should appear bright and distinct. The 28S band is usually twice as intense as the 18S band. Smaller, diffuse bands may represent tRNA or 5S rRNA. A faint smear between 18S and 28S could indicate mRNA or other RNA fragments. If DNA contamination is present, it may appear as a higher molecular weight smear. Degraded RNA will show a smeared or fragmented pattern. Capture images using a digital camera.

3. cDNA synthesis

Fluorescence quantitative PCR experimental procedure

1. Reaction system:

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 6,000 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 transfer the tube to an ice-water bath to cool both inside and outside, then add 0.5 μl of MMLV enzyme. Incubate at 37°C for 60 minutes.

3. After incubation, heat the mixture at 95°C for 3 minutes in a dry bath to terminate the reaction. The resulting product is cDNA, which should be stored at -80°C until further use.