Real time fluorescence quantitative PCR (experimental service)

1. Service Overview

Real time fluorescence quantitative PCR (experimental service)Quantitative Real time PCR (qPCR) refers to the method of adding fluorescent groups to the PCR amplification reaction system, using fluorescence signal accumulation to monitor the entire PCR process in real time, and finally quantitatively analyzing unknown templates through standard curves. As the "gold standard" technology in molecular biology research, qPCR has become a tool for gene expression analysis, pathogen detection, and genetic variation research due to its sensitivity, specificity, and accuracy.

We rely on the PCR detection platform to provideSample nucleic acid extraction, reverse transcription, qPCR amplification to data analysisOne stop technical service. Whether you need to detect the expression levels of mRNA, miRNA, lncRNA, or perform DNA copy number variation (CNV) analysis, we can provide you with high-quality data that meets the standards.

2. Technical principles and methods

We offer two mainstream detection strategies to meet different experimental needs:

• SYBR Green I dye method:

• Principle:SYBR Green I dye can specifically bind to the small grooves of double stranded DNA, and the fluorescence signal is enhanced after binding. With the increase of PCR products, the fluorescence signal synchronously enhances.

• Applicable to:Initial screening of gene expression differences, large-scale sample testing, and cost sensitive projects. We cooperate with the melt curve analysis to ensure the singularity of the amplified product.

• TaqMan probe method:

• Principle:Using specific probes designed for target sequences (5 'end labeled with fluorescent reporter groups, 3' end labeled with quenching groups). When the probe is intact, there is no fluorescence. During the amplification process, the hydrolysis of Taq enzyme cleaves the fluorescent group and emits fluorescence.

• Applicable to:High specificity experiments, multiplex PCR detection, SNP typing, and micro sample detection are required.

3. Service Content and Entire Process

We strictly follow standardized operating procedures (SOP) to ensure precise and controllable control of each step:

Stage 1: Experimental Design and Optimization

• Primer/probe design:Design cross intron primers targeting the target gene to avoid interference from genomic DNA contamination; Screening for reference genes (Housekeeping Genes, such as GAPDH, ACTB, 18S rRNA, etc.).

• Pre experimental verification:The primer specificity was verified by ordinary PCR and agarose gel electrophoresis, and the annealing temperature was optimized.

Phase 2: Sample Processing

• Nucleic acid extraction:For different sample types (cells, tissues, blood, exosomes, etc.), high-quality total RNA or DNA is extracted using specialized reagent kits for concentration and purity (OD260/280) detection and integrity evaluation.

• Reverse transcription (cDNA synthesis):Use Oligo (dT) or random primers for mRNA; Use stem loop primers or tail addition method for miRNA to ensure uniform reverse transcription efficiency.

Phase Three: On board Inspection

• System construction:Configure a high fidelity qPCR reaction system with at least 3 technical replicates to eliminate sample errors.

• Program operation:Real time collection of fluorescence signals and generation of amplification curves.

Stage Four: Data Analysis

• Relative quantification:adopt2−ΔΔCtCalculate the fold change of gene expression differences between the experimental group and the control group using the method.

  
  

• Quantitative:Construct plasmid standards, draw standard curves, and calculate the exact copy number of the target gene in the sample.

  
  

4. Our core strengths

1. Hardware platform:Mainstream quantitative PCR instruments have extremely small inter well differences and high data stability.

2. Strict quality control system:Following international standards MIQE（Minimum Information for Publication of Quantitative Real-Time PCR Experiments） Guide. Strictly monitor amplification efficiency (90% -110%) and linear relationship (R ²>0.99) to prevent false positives/false negatives.

3. Complex sample processing:Having rich experience in FFPE slicing, micro tissue, extracellular vesicle and whole blood sample processing, effectively removing PCR inhibitors.

4. Professional data delivery:Not only does it provide raw Ct values, but it also offers amplification curves, melting curves, relative expression bar charts/heat maps, and generates a complete report containing detailed experimental materials and methods, directly meeting the requirements for SCI publication.

5. Application scenarios

• Gene expression level analysis:Differential expression validation of mRNA, lncRNA, and circRNA under different treatment conditions or disease models (qPCR validation after RNA seq).

• MiRNA detection:Research on the expression regulation of microRNAs.

• DNA copy number variation (CNV):Tumor gene amplification or deletion detection, transgenic copy number identification.

• SNP typing validation:Genotyping identification of single nucleotide polymorphism sites.

• Detection of pathogenic microorganisms:Virus load determination and bacterial qualitative and quantitative analysis.

• ChIP-qPCR：Enrichment validation of DNA fragments after chromatin immunoprecipitation and study of protein DNA interactions.

6. Sample submission requirements and delivery standards

• Sample submission suggestion:

• Cells: Cell precipitation>

• 1*10^6， Wash with PBS and freeze quickly.

• organizationAnimal tissue>50mg, plant tissue>100mg, liquid nitrogen quick freezing or RNAlater preservation

• Blood:Whole blood needs to be anticoagulated or directly provided with separated serum/plasma.

• RNA/DNA：Concentration>50ng/μ L, total amount>1 μ g, dry ice transportation.

• Delivery content:Complete experimental report (including experimental steps and reagent information), raw data files (. eds/. xls), and data analysis result charts (Excel statistical tables and high-definition images).