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Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Related Experiment Video

Updated: Aug 9, 2025

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
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Quantitative PCR of Small Nucleic Acids: Size Matters.

Jay Min Lim1, Rahul Tevatia1, Ravi F Saraf2

  • 1Vajra Instruments Inc., 8300 Cody Drive, Ste C, Lincoln, NE 68512, USA.

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|February 23, 2023
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Summary
This summary is machine-generated.

Quantitative PCR (qPCR) struggles to precisely measure low concentrations of small nucleic acids (NA) like microRNA (miRNA). The study finds that PCR product length significantly limits the quantification accuracy for these important disease biomarkers.

Keywords:
IntercalationsMicroRNANucleic acidsPolymerase chain reactionReverse transcription

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Area of Science:

  • Biomarker Discovery
  • Molecular Diagnostics
  • Quantitative Biology

Background:

  • Small nucleic acids (NA), including microRNA (miRNA), are promising biomarkers found in minimally invasive samples.
  • Quantitative PCR (qPCR) is the gold standard for measuring nucleic acid copy numbers.

Purpose of the Study:

  • To investigate the impact of poly(A) ligation and small NA size on the limit of quantification (LOQ) in qPCR.
  • To identify factors limiting the accurate quantification of small NA using qPCR.

Main Methods:

  • Quantitative PCR (qPCR) was used to measure small nucleic acids.
  • The effect of poly(A) ligation during cDNA synthesis was evaluated.
  • The relationship between cycle threshold (Ct) and nucleic acid concentration ([c]) was analyzed.

Main Results:

  • Small NA exhibit a sharp transition in cycle threshold (Ct) values.
  • While the limit of detection (LOD) can be in the femtomolar range, the LOQ is reduced by over three orders of magnitude, reaching the picomolar range.
  • PCR product length was identified as the primary factor limiting the LOQ for small NA.

Conclusions:

  • The LOQ of qPCR is significantly reduced for small NA compared to its LOD.
  • PCR product length is a critical consideration for optimizing primer design and improving qPCR accuracy for small NA quantification.
  • Accurate quantification of small NA biomarkers requires careful attention to assay design limitations.