Real-Time PCR Overview

Real-Time PCR

• Also known as Quantitative PCR (qPCR)
• Offers high sensitivity
• Has a long dynamic range
• Results expressed as numbers
• Automated
• Not size-based discrimination or post-PCR processing needed.
• Uses fluorescent dyes (e.g. SYBR Green) or probes (e.g. TaqMan)

Conventional PCR vs. Real-Time PCR

• End-Point Conventional PCR:
  • Low sensitivity
  • Short dynamic range (< 2 logs)
  • Non-automated
  • Size-based discrimination only
  • Results not expressed as numbers (not quantitative)
  • Ethidium bromide staining not very quantitative
  • Low resolution, poor precision
  • Post-PCR processing required
• Real-Time PCR:
  • High sensitivity
  • Long dynamic range
  • Automated
  • Not size-based discrimination
  • Results expressed as numbers (quantitative)
  • Fluorescent dyes or probes for quantitative analysis
  • No post-PCR processing needed

RT-PCR vs. Serology

• Real-Time PCR (RT-PCR):
  • More accurate for SARS-CoV-2, CMV, Brucella, Leishmania, and Toxoplasma detection
• Serology test:
  • Another method for detecting viruses
  • Not always best suited for all cases; different tests are better for different viruses.

RT-PCR Types

• qPCR: Real-Time/Quantitative Polymerase Chain Reaction
• RT-PCR: Reverse Transcription Polymerase Chain Reaction
• qRT-PCR: qPCR and RT-PCR combined technique (e.g., SARS-CoV-2 [COVID-19])

Reverse Transcriptase PCR (RT-PCR) - cDNA Synthesis

• Variation of Polymerase Chain Reaction (PCR)
• Amplifies target RNA
• Addition of reverse transcriptase (RT) enzyme
• Converts RNA to cDNA
• PCR amplification follows for detection of RNA targets

cDNA Synthesis Protocols

• Mix components, vortex briefly, and spin down
• Prepare reaction with specific components
• Use specific primers (e.g., random primer mix)
• Complete process using specific cycle steps/ temperatures/ times

RT-qPCR

• Real-time reverse transcription polymerase chain reaction
• Addresses the need for quantification
• Used in molecular medicine, biotechnology, microbiology, and diagnostics.
• Quantifies mRNA as transcriptional biomarkers in molecular diagnostics, normal biological processes, and pathogenic and pharmacological reactions to therapeutic interventions.

Real-time PCR Applications

• Diagnostics (detecting viruses, bacteria, etc.)
• Gene expression (quantifying mRNA levels)

qPCR Quantification Types

• Absolute: Needs serial standard concentrations of control DNA, plasmid, or RNA for accurate quantity determination
• Relative: Uses reference genes (housekeeping genes) to normalize and estimate changes in gene expression compared to a control. (e.g., ACT, a common reference gene).

qPCR Assays

• SYBR Green qPCR: Uses SYBR Green dye that intercalates with double-stranded DNA. Intensity is measured during PCR cycles
• TaqMan Probe qPCR: Uses DNA probes (tagged with a fluorescent reporter at one end and quencher at the other end). The quencher prevents reporter dye fluorescence when intact. Cleavage of the probe releases the reporter dye from the quencher; increases fluorescence as cycles progress.

High Resolution Melt (HRM) - qPCR

• A method used for DNA melting analysis of PCR products.
• Used to assess differences in melting temperatures between samples
• Identifies sequence variations, including mutations, for identifying DNA variations.

Other Methods and Applications

• Detecting bacteria (e.g. Mycobacterium tuberculosis, M. bovis)
• Diagnosing leishmaniasis
• Detecting copy number variants (CNVs) e.g. spinal muscular atrophy (SMA), cystic fibrosis (CF) and human breast carcinoma (HER-2/neu).
• Digital PCR: Used to quantify nucleic acids. (DD-PCR)
• Detecting different pathogens or other disease states
• Real-time PCR for predicting virologic response to IFN therapy. Viral load of Hepatitis B and other uses
• Detecting cancer-related mutations. Detecting thyroid cancer biomarkers