What would be the usefulness of quantifying cDNA before conducting a PCR, and how could it influence the effectiveness and reproducibility of the obtained results?
not much use most of the time. NTPs and primers will absorb in the UV and purifying will lose valuable material and even tiny amounts of cDNA will amplify using pcr so often it is best to just go directly to the amplification step
I always quantified the RNA used for synthesizing cDNA. This practice ensures accuracy in determining the amount of cDNA produced. Essentially, the quantity of cDNA is directly linked to the initial amount of RNA utilized in the synthesis process. Therefore, quantifying the RNA beforehand serves as a crucial step in ensuring the reliability and reproducibility of downstream analyses involving cDNA.
We do not directly quantify cDNA in PCR experiments. Instead, we measure the RNA template and then reverse transcribe it to produce cDNA. After synthesis, cDNA is usually utilized as a template for quantifiable PCR amplification. To guarantee that the same amounts of RNA are used to create the same amounts of cDNA, or one can dilute the cDNA to use the same amounts in the real-time PCR experiment.
To ensure that cDNA that has been synthesised from RNA does not have contamination, we do run a contamination panel before using it for the designated purpose.
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