Real time RT PCR

DNA contamination

Removal of DNA contamination

Contamination of RNA samples with trace amounts of genomic DNA can interfere with real-time RT-PCR quantification if the PCR primers used are also able to amplify genomic DNA sequences. To avoid the negative effects of genomic DNA contamination, careful primer design is required (see Primer design). If this is not possible, RNA samples should be treated with DNase I to digest contaminating DNA.

Detecting DNA contamination in RT-PCR

Use of appropriate controls will enable the detection of any contaminating DNA in the RT-PCR. Reactions should be set up with and without the reverse transcriptase. The presence of a product in the absence of the reverse transcriptase indicates contamination.

- Introduction
- Polymerase chain reaction (PCR)
- Types of PCR
- PCR primer design
- PCR conditions | Primer annealing specificity | PCR buffers
- Degenerate primers: Design and use
  - Primer sequence:
  - Primer concentration:
- PCR amplification: Long PCR products
- DNA polymerases used in PCR
- PCR cycle steps
- Commonly used terms in PCR
- Commonly used terms in PCR
- PCR quantification
- Absolute Quantification qPCR | Relative Quantification qPCR
  - What is absolute quantification?
  - What is relative quantification?
- PCR efficiency
- Real time PCR
- Real-time polymerase chain reaction
  - What is SYBR Green I in PCR?
  - PCR probes
- Real time RT PCR
- RT-PCR | Reverse transcription PCR
- DNA contamination
  - Removal of DNA contamination
  - Detecting DNA contamination in RT-PCR
- Digital PCR
- Fundamentals of digital PCR
- dPCR vs qPCR vs end-point PCR
- Comparison of digital PCR methods
- Reference Genes and Controls
- Endogenous reference genes | Housekeeping genes
- PCR controls
- References