Question

In RT-qPCR, which of the following is measured in real time?

1) the amount of RNA used as a template during the RT step
2) the amount of DNA produced during the PCR step
3) the amount of cDNA produced during the RT step

Answer 1

During RT-qPCR, the real-time measurement pertains to the amount of DNA produced in the PCR step, using fluorescence to quantify the original target sequence.

Step-by-step explanation:

In RT-qPCR, the measurement taken in real-time is the amount of DNA produced during the PCR step. This is done using a fluorescent dye that binds to the double-stranded DNA as it is synthesized, allowing the quantification of the original target sequence.

Unlike the initial reverse transcription (RT) step where RNA is converted into cDNA, the real-time quantification occurs during the PCR cycles as DNA is amplified from the cDNA template.

Answer 2

The measurement in RT-qPCR is of the amount of DNA produced during the PCR step, monitored by the increase in a fluorescent signal that correlates with the quantity of double-stranded DNA produced. So, the correct option is 2) the amount of DNA produced during the PCR step.

Step-by-step explanation:

In RT-qPCR, the measured parameter in real-time is the amount of DNA produced during the PCR step. This technique involves converting mRNA to complementary DNA (cDNA) using reverse transcriptase (RT), in a process known as reverse transcription. Then, by applying PCR, this cDNA is amplified and quantified. The use of fluorescent markers in RT-qPCR allows for the monitoring of the amplification process in real time, providing a quantitative measure of how many original template molecules were present in the sample at the start of PCR. This is because the fluorescent signal increases proportionally as the amount of double-stranded DNA produced during PCR increases, thus facilitating the quantitation of the original target sequence.

Applications of RT-qPCR are extensive and include detecting gene expression levels, measuring viral loads in clinical settings, and various research purposes. By accurately measuring the amount of double-stranded DNA created during the PCR steps, researchers and clinicians can determine the number of DNA copies or organisms present in the sample, which can reflect the levels of gene expression or the viral load in patients.