qPCR (real-time PCR) protocol explained

A comprehensive overview of quantitative Polymerase Chain Reaction (qPCR), also known as real-time PCR. This technique is widely used in laboratories for detecting and quantifying DNA and RNA sequences, playing a crucial role in various applications, including gene expression analysis and pathogen detection.

Key points covered in the video include:

1. Principle of qPCR: The method amplifies specific DNA sequences and measures the amount of amplified product in real-time using fluorescent dyes or probes.
2. Components of qPCR:
   • Template DNA/RNA: The target sequence to be amplified.
   • Primers: Short DNA sequences that initiate the DNA synthesis.
   • DNA Polymerase: An enzyme that extends the primers to form the new DNA strands.
   • Fluorescent Probes/Dyes: Molecules that emit fluorescence when bound to the amplified DNA, allowing quantification.
3. qPCR Workflow:
   • Sample Preparation: Extraction of nucleic acids from the sample.
   • Reaction Setup: Mixing of template, primers, polymerase, and fluorescent probes/dyes.
   • Thermal Cycling: Repeating cycles of denaturation, annealing, and extension to amplify the target sequence.
   • Data Collection: Monitoring fluorescence intensity during each cycle to quantify the DNA.
4. Data Analysis:
   • Threshold Cycle (Ct): The cycle number at which fluorescence exceeds a predefined threshold, inversely related to the initial amount of target nucleic acid.
   • Quantification Methods: Standard curve and comparative Ct methods to determine the quantity of the target sequence.

The video emphasizes the importance of proper experimental design, including the selection of appropriate controls and reference genes, to ensure accurate and reliable qPCR results.