10.3.4. Controls

As for strand-specific RT-PCR, MLPA requires a large number of controls to rule out the possibility of artefactual results due to the methods used.

  • A nucleic acid-free control.
    - The two half-probes should not be able to ligate without a template.
  • A reverse transcriptase-free control.
    - The two half-probes should not be able to ligate on an RNA template.
  • A cDNA primer-free control.
    - Identifies possible self-priming of RNA for cDNA synthesis.
  • A half-probes-free control.
    - Only the two half-probes should generate an amplifiable template for PCR.
  • A ligase-free control.
    - The two half-probes should not be able to ligate without ligase.
  • A PCR primer-free control.
    - The two half-probes should not be able to function as PCR primers either with each other or with the cDNA primers.