Molecular techniques: real-time PCR interpretation: practice question

A Biochemistry question in the style of the NEET PG paper. Answer it first, then read why the right option is right and why each of the others is wrong.

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Biochemistry · Molecular techniques: real-time PCR interpretation

Sample item

In a quantitative reverse transcription PCR for HIV-1 RNA, sample A crosses the fluorescence threshold at cycle 20 and sample B at cycle 28. Assuming equal efficiency of 100% in both reactions, how do the starting template amounts compare?

AThe amounts are equal because both crossed the thresholdBSample A has about 256 times more starting RNA than sample BCSample B has about 256 times more starting RNA than sample ADSample A has about 8 times more starting RNA than sample B

Original practice item, not a recalled exam question. Nothing here is recorded.

The answer and the reasoning

B. Sample A has about 256 times more starting RNA than sample B

Reverse transcriptase first copies RNA into cDNA, and the amplification is then followed by fluorescence. The cycle threshold (Ct) is the cycle at which signal passes the threshold, and a lower Ct means more starting template. With doubling each cycle, the difference of 8 cycles gives 2 to the power 8, which is 256-fold.

Option by option

A. The amounts are equal because both crossed the threshold
The threshold is crossed at different cycles because the starting amounts differ.
B. Sample A has about 256 times more starting RNA than sample B (correct)
A needs 8 fewer doublings, so it began with 2^8 = 256 times more template.
C. Sample B has about 256 times more starting RNA than sample A
The relationship is reversed here: a higher Ct means less starting template.
D. Sample A has about 8 times more starting RNA than sample B
Eight is the difference in cycles, and the fold change is 2 raised to that power, not 8.

Source note: Original NextPrep sample item — pending expert review. This item is original practice material awaiting independent expert review, so confirm important facts in a standard textbook.

Questions about this Biochemistry question

What is the answer to this Biochemistry question?

The best answer is B, Sample A has about 256 times more starting RNA than sample B. Reverse transcriptase first copies RNA into cDNA, and the amplification is then followed by fluorescence.

Why is "The amounts are equal because both crossed the threshold" not the answer?

The threshold is crossed at different cycles because the starting amounts differ.

Why is "Sample B has about 256 times more starting RNA than sample A" not the answer?

The relationship is reversed here: a higher Ct means less starting template.

Why is "Sample A has about 8 times more starting RNA than sample B" not the answer?

Eight is the difference in cycles, and the fold change is 2 raised to that power, not 8.

What concept does this question test?

It tests Molecular techniques: real-time PCR interpretation in Biochemistry. The cycle threshold (Ct) is the cycle at which signal passes the threshold, and a lower Ct means more starting template. With doubling each cycle, the difference of 8 cycles gives 2 to the power 8, which is 256-fold.

How hard is this question?

We tag it as a multi-step question: it asks you to combine more than one idea. The tag is our own editorial judgement, not an official difficulty rating, so read the reason for every option, not only the answer.

How should I study Molecular techniques: real-time PCR interpretation?

Build one table of enzyme deficiency, disease, inheritance and key finding, then revise it by spaced recall. Then use the flashcards and the other Biochemistry questions on this site to test what stuck.

Is this a real NEET PG question?

No. It is an original practice item (editorial status: sample, version 1). NextPrep does not publish recalled or leaked questions, and independent faculty review of this item has not been completed yet, so confirm important facts in a standard textbook.