What are the disadvantages of end-point RT-PCR?
Posted November 9, 2023
End-point RT-PCR analysis, which is based on the plateau phase of PCR reaction, is used to analyze amplified products after all the cycles of the PCR reaction have been completed. In this method, amplicons are separated by agarose gel electrophoresis and visualized using a DNA binding dye, such as ethidium bromide (EtBr), to determine the size of the DNA molecules in the range of 500 to 30,000 bp. While endpoint RT-PCR has its advantages, it also comes with some disadvantages:
- Poor precision and low sensitivity: In general, endpoint RT-PCR may be less sensitive than real-time PCR methods, as it relies on the final PCR product's detection rather than the continuous monitoring of amplification during the reaction.
- Short dynamic range < 2 logs: endpoint RT-PCR has a limited dynamic range. This limitation can be problematic when analyzing samples with varying RNA levels.
- Results are not expressed as numbers: One of the primary drawbacks of endpoint RT-PCR is its inability to provide real-time quantitative data. It only confirms the presence or absence of a specific RNA sequence but doesn't quantify the amount of RNA in the sample.
- Post-PCR processing: To analyze the results of endpoint RT-PCR, additional steps such as gel electrophoresis are often necessary. This adds extra time and complexity to the experimental workflow.
- Low resolution, size-based discrimination only
- Not suitable for automation