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Quantitative RT-PCR or RT-qPCR is a highly efficient means of quantifying the levels of individual transcripts....

Quantitative RT-PCR or RT-qPCR is a highly efficient means of quantifying the levels of individual transcripts. Which of the following primers is critical to carry out the first step in RT-qPCR?

-dGGGGGG (poly dG)

-dCCCCCC (poly dC)

-TTTTTTT (poly T)

-dAAAAAA (poly dA)

-none of them

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Answer #1

TTTTTTT (POLY T)

Advantages Disadvantages
One-step
  • Less experimental variation since both reactions take place in the same tube
  • Fewer pipetting steps reduces risk of contamination
  • Suitable for high throughput amplification/screening
  • Fast and highly reproducible
  • Impossible to optimize the two reactions separately
  • Less sensitive than two-step because the reaction conditions are a compromise between the two combined reactions
  • Detection of fewer targets per sample
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