PCR and Transcriptome Analysis Techniques

Posted on Apr 3, 2025 in Biology

Primers

  • Length: 18-30 bp
  • Avoid self-complementarity
  • Flank regions 100-1000 bp apart
  • Multiplexing with multiple primers is possible
  • 3’ end should not be GC-rich and avoid complementarity to prevent nonspecific binding and primer dimers

Taq Polymerase

  • Source: Taquaticus
  • Optimal efficiency: 75-80°C
  • Replication speed: ~1 kb in <10 seconds

QRT-PCR

  • Mostly semi-quantitative
  • Fluorescence probe with reader enables readout
  • Increase in fluorescence indicates starting material
  • dsDNA dye like SYBR Green is not quantitative
    • Shows relative expression; only binds to dsDNA
    • Can estimate the number of different products formed
    • Requires DNA melting, primer addition, and dsDNA synthesis
    • Easy setup, inexpensive, good qualitative data, but cannot multiplex due to single color
  • Fluorescent Quantification: Fluorescent probes specific to target DNA
    • Specific Probe Type I (Primer)
      • Probe bound with a quencher and a reporter
      • Upon binding to ssDNA, quencher and reporter separate, allowing reporter fluorescence
      • Fluorescence increases with each reaction cycle as more probes bind
      • Hairpin structure prevents fluorescence
      • Primer binds only once per target, ensuring quantitative measurement
      • High threshold and background fluorescence due to hairpin unfolding in solution (salt concentration). Use manufacturer’s buffer.
    • Specific Probe Type II
      • Probe with quencher and reporter binds to ssDNA
      • Polymerase degrades probe during replication, releasing reporter and enabling fluorescence
      • Low background
      • High capacity
      • More accurate per copy number
    • Determining Starting Copy Number:
      • Generate a PCR reaction curve (standard curve)
      • Starting copy number is initially masked by background
      • Quantitative on gel
  • Absolute vs. Relative Quantification:
    • Relative quantification is more common (99% of PCR)
    • Absolute Quantification:
      • Uses TaqMan with a standard curve of known copy numbers
      • Standards must be amplified with the same primers as the sample, ensuring equal amplification efficiency
    • Relative Quantification:
      • Gene expression levels are quantified as a ratio between the target gene and an endogenous reference gene
      • Include positive and negative controls

Transcriptome Analysis

  • Analysis of all RNA
  • Expensive
  • Targets poly-A tail for expressed genes
  • Process: mRNA extraction -> reverse transcription, fluorescent labeling -> combine equal amounts and hybridize -> scan
  • Color coding: Green (control), Red (experimental), Yellow (both)
  • Software analyzes data
  • Challenges: Alternative splicing leads to cell-specific mRNA variation
  • Requires sequencing all molecules to avoid translation initiation at different RNA sites
  • mRNA size variation due to poly-A tail site choice regulated by external signals

Next Generation Sequencing

  • RNA sequencing provides data, but proteome analysis is needed to understand cell phenotype

Whole Genome Array

  • Contains oligos for every gene