RT-PCR and End Point PCR Notes

Module Learning Objectives

  • Discuss and interpret experimental evidence supporting key concepts.

Workshop Learning Objectives

  • Discuss different levels of gene expression.
  • Describe RT-PCR.
  • Describe and interpret end-point PCR data.

Levels of Gene Expression

  • Central Dogma: DNA makes RNA (or more DNA), RNA makes protein.
    • Reference: Crick, F.H.C. (1958): On Protein Synthesis. Symp. Soc. Exp. Biol. XII, 139-163

Gene Expression

  • Gene expression can be discussed in terms of protein amount or mRNA amount.
  • Protein and mRNA levels may not always correlate.
    • Checking both is essential for research.
  • Stability of mRNA or protein plays a key role.
    * Example: HIF1α is expressed as mRNA, but the protein is broken down in oxygen (Video 5).

Workshop Learning Objectives

  • Discuss the process for generating a cDNA library.
  • Discuss application of end-point PCR.
  • Interpret end-point PCR data.

RT-PCR

  • Most molecular biology methods work on DNA, not RNA.
  • mRNA is converted to cDNA.
  • cDNA is the complement to mRNA.
  • cDNA exists after splicing in eukaryotes and lacks introns or other genomic information.
  • cDNA contains only what mRNA expresses in the cell.

RT-PCR Details

  • A poly-T primer complements the poly-A tail of eukaryote mRNA.
    • RNA without a poly-A tail is not converted into cDNA.
    • For other types of RNA (e.g., prokaryotic), use a different primer such as a random hexamer.

RT-PCR - Key Questions

  • Why convert mRNA to cDNA in molecular biology experiments?
  • What is the significance of RNA processing in eukaryotic cells and its impact on cDNA composition?

RT-PCR - cDNA Significance

  • What information does cDNA exclusively represent?
    • How does this make it a valuable tool in studying gene expression?
  • Examples of using cDNA to investigate cellular processes.

End Point PCR

  • Polymerase Chain Reaction (PCR)

    1. Denaturation at 95-96°C.
    2. Annealing at 68°C.
    3. Elongation at 72°C.

End Point PCR - Steps

  1. Denaturation
    • Double-stranded DNA template is heated to 94-98°C.
      • Strands separate, breaking hydrogen bonds.
      • Forms two single-stranded DNA molecules.
  2. Primer Annealing
    • Temperature lowered to 50-65°C.
      • DNA primers bind (anneal) to single-stranded DNA at specific locations (sense and antisense).
      • Primers flank the target DNA sequence for amplification.

End Point PCR - Steps Continued

  1. Extension (Elongation)
    • Temperature raised to 72-75°C.
      • DNA polymerase synthesizes a new DNA strand.
      • Adds nucleotides complementary to the template strand.
      • Extends primers and produces a copy of the target DNA sequence.
  2. Repeated Cycles
    • Denaturation, annealing, and extension are repeated.
      • Each cycle doubles the amount of DNA.
      • Newly synthesized strands become templates for subsequent cycles.
      • The number of cycles determines the final amplified DNA amount.

End Point PCR - Gel Analysis

  1. Load DNA Sample
    • Load PCR product of gene of interest.
    • Load control PCR products and controls.
      • No reverse transcriptase control (no template cDNA).
      • Positive control for a known gene.
      • Molecular weight marker to check PCR product length.
  2. Apply Current
    • Submerge gel in buffer solution.
      • Apply electric current across the gel.
      • Negatively charged DNA moves toward the positive electrode.
      • Separation is size-based through the agarose.
  3. Analyze the Gel
    • Stain gel with dye that binds to DNA.
      • Visualize DNA bands under UV light.
      • Compare migrated DNA bands with a molecular weight ladder (DNA fragments of known sizes).
      • Ladder estimates the size of amplified DNA fragments.
      • Bands represent DNA presence and their position indicates size.

End Point PCR - Key Questions

  • Why is primer annealing essential in PCR?
    • What is the role of DNA primers and the temperature range during primer annealing?
  • How do researchers design primers for specificity?
    • Bioinformatics tools analyze the target gene, avoiding regions prone to secondary structures or cross-hybridization.

End Point PCR - Controls and Markers

  • Describe the purpose of loading a molecular weight marker in gel electrophoresis.
    • How does the use of a molecular weight ladder aid in the interpretation of PCR results?
  • Explain why loading controls (no reverse transcriptase control, positive control) are essential.
    • What information do these controls provide?
    • How do they help ensure the reliability of experimental outcomes?

End Point PCR - Gel Example

  • Molecular weight ladder confirms the PCR product size.
    • Note: This is the size of the amplified fragment, not the total gene size.
  • Two conditions: with or without FGF2 stimulation.
    • Brighter band for FGF2 stimulated cells.
  • GAPDH +ve control band: GAPDH is a 'housekeeping gene' and usually expressed.
  • No band in the 'No RT' sample: without reverse transcriptase, no cDNA can be made.

End Point PCR - Additional Notes

  • Older papers may have cropped gels with missing controls and ladders due to space limitations.
  • Today, full uncropped images are expected but not always provided.

End Point PCR - HPV Example

  • RT-PCR for HPV16 E6 and E7 gene expression.
    • Gel shows PCR products from cDNA synthesized from HPV positive UBC, a negative DNA control, and HeLa and UM-SCC-47 cells.
  • Does the image provided show evidence that the patient was infected with human papillomavirus?