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Dye-based quantitative PCR (Quantitative PCR, qPCR) is a core technology in molecular biology for the detection and quantification of target nucleic acid sequences. It is widely used in gene expression analysis, pathogen detection, SNP genotyping, copy number variation analysis and other fields. Its principle is based on the incorporation of fluorescent dyes (such as SYBR Green I) by DNA polymerase during the extension process. The dye emits fluorescent signals after binding to double-stranded DNA, and the fluorescence intensity is proportional to the accumulation of PCR products, so as to realize real-time monitoring and accurate quantification of the initial template amount.

1. RNA Extraction and Quality Control

High-quality RNA is the prerequisite for accurate quantification. The extraction of RNA should generally follow the following principles:

① Use RNase-free consumables and reagents, and avoid RNase contamination during extraction;

② Ideal total RNA should show clear 28S and 18S rRNA bands in agarose gel electrophoresis imaging, with a brightness ratio of about 2:1;

③ Determine the A260/A280 ratio by spectrophotometer. Pure RNA should be in the range of 1.8–2.1; the A260/A230 ratio should be greater than 1.5 to exclude contamination by organic solvents or salts;

④ RNA samples should be subjected to reverse transcription as soon as possible, or stored at -80°C to avoid degradation.

2. cDNA Synthesis by Reverse Transcription

Using mRNA as the template, reverse transcriptase is used to synthesize complementary DNA (cDNA), which serves as the template for subsequent qPCR.

Select oligo(dT) primers or gene-specific primers according to experimental requirements. The former is suitable for mRNA with poly(A) tails, random hexamer primers are suitable for RNA without poly(A) tails or prokaryotic RNA; the latter is used for targeted reverse transcription of specific transcripts. Conventional reverse transcriptases generally react at 42–50°C, and thermostable reverse transcriptases can work at up to 55°C or higher to overcome the hindrance of RNA secondary structures.

3. Primer Design

The specificity and amplification efficiency of primers directly determine the reliability of quantitative results. The following principles are usually followed in primer design:

① The length of amplified products is controlled at 80–200 bp. Too long products will reduce amplification efficiency, while too short products make it difficult to distinguish specific products from non-specific products through melting curves;

② The Tm difference between upstream and downstream primers shall not exceed 2°C;

③ Avoid three or more consecutive G/C bases at the 3' end of primers to reduce non-specific amplification;

④ Primers should be designed to span introns or be located at the junction of different exons, or span long introns on genomic DNA, so as to eliminate the interference of genomic DNA contamination on quantitative results;

⑤ Verify primer specificity by conventional PCR before formal experiments to ensure a single clear band.

4. Quantitative PCR Reaction

Dye-based qPCR uses fluorescent dyes such as SYBR Green I to intercalate into double-stranded DNA and emit light. The accumulation of products is reflected by real-time monitoring of fluorescence signal intensity.

The following principles shall be followed when preparing a 20 μL qPCR reaction system:

① The addition amount of template cDNA should be appropriate. Generally, the reverse transcription product is diluted 5–10 times, and 1–2 μL is taken, so as to avoid inhibitory components in high-concentration templates affecting amplification efficiency;

② The final concentration of primers is usually 0.2–0.4 μM. Excessively high concentration easily leads to primer dimer formation, while excessively low concentration reduces amplification efficiency;

③ The cycling program is usually set as: pre-denaturation at 95°C for 30 s to 3 min; denaturation at 95°C for 10–15 s, annealing/extension at 60°C for 30–60 s, for a total of 40 cycles; melting curve analysis from 60°C slowly rising to 95°C.

The amplification efficiency (Efficiency) should be within 90%–110% (corresponding to a standard curve slope of approximately -3.1 to -3.6), and the correlation coefficient R² should be greater than 0.98. The melting curve should show a single sharp peak. If multiple peaks or shoulder peaks appear, it indicates non-specific amplification or primer dimers.

5. Data Analysis and Quality Control

The reliability of qPCR data depends on rigorous reference gene selection and quality control standards.

The following principles are usually followed:

① Select reference genes with stable __expression (such as GAPDH, β-actin, 18S rRNA, etc.). Multiple reference genes can be used for normalization when necessary;

② The relative quantification method (2^-ΔΔCt method) is used to compare gene expression differences among different samples, which requires that the amplification efficiencies of target genes and reference genes are close;

③ Set at least three technical replicates for each sample, and the standard deviation of Ct values should be less than 0.5;

④ Set no-template control (NTC) to detect system contamination, and set no-reverse transcription control (-RT) to exclude genomic DNA contamination.