Question

Real time PCR: how is it analyzed? what can be done to improve real time PCR? How do you know if the bullfight was good or not? What does it mean if the real time parameters are high, low, or if there were faults in something in the process seeing the results? Abound in your response.

Real time pcr:
• How to do?
• How it is interpreted?
• How it is optimized

Answer 1

• Real-time PCR (Polymerase Chain Reaction) employs the use of the fluorescent signal which is generated by either an intercalating dye or via the breakdown of the dye labelled probe which happens duuring the amplification of the required or targeted sequence. The intensityof the sinal generated is used to detect the amount of the nucleic acid present in the sample (quantification of the DNA).

• The results of the real-time PCR are plotted on a graph, where the intensity of the fluorescence is depicted on the Y-axis while the number of the PCR cycles are on the X-axis. This amplification plot is studied and interpreted to calculated the amount of DNA present in the sample.

• The real-time PCR technique can be optimized by screening the primers used using the gel electrophoresis. Moreover, the dilution of the DNA sample (based on the number of copies of the concerned gene) should be done. The amplicon should be targetted towards the 3' end of the transcript (in case of reverse transcription).