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Related Concept Videos

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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Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
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An improved method for detecting circulating microRNAs with S-Poly(T) Plus real-time PCR.

Yanqin Niu1,2, Limin Zhang1, Huiling Qiu1,2

  • 1Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China.

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We developed S-Poly(T) Plus, a simple and sensitive real-time PCR assay for analyzing circulating microRNAs (miRNAs). This method enhances miRNA detection in serum and plasma for disease diagnosis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Circulating microRNAs (miRNAs) are valuable biomarkers for disease diagnosis.
  • Existing methods for miRNA analysis often lack sufficient sensitivity or simplicity.
  • Accurate detection of circulating miRNAs is crucial for advancing personalized medicine.

Purpose of the Study:

  • To develop a simple, sensitive, and specific method for the analysis of circulating miRNAs.
  • To improve upon the previously developed S-Poly(T) method for enhanced miRNA detection.
  • To validate the new method using patient samples with pulmonary arterial hypertension.

Main Methods:

  • Development of the S-Poly(T) Plus real-time PCR assay, integrating polyadenylation and reverse-transcription in a single step.
  • Optimization of total RNA isolation from serum/plasma (S/P miRsol) using glycogen to maximize RNA yield.
  • Quantification of miRNA expression profiles in serum samples from patients with pulmonary arterial hypertension associated with congenital heart disease.

Main Results:

  • The S-Poly(T) Plus assay demonstrated high sensitivity and specificity for circulating miRNA detection.
  • The S/P miRsol method significantly improved RNA yield from serum/plasma samples.
  • The method successfully quantified miRNA expression profiles in patient sera, enabling biomarker discovery.
  • The assay can measure 266 different miRNAs from a small volume (100 μl) of serum or plasma.

Conclusions:

  • The S-Poly(T) Plus real-time PCR assay is a simple, sensitive, and specific tool for circulating miRNA analysis.
  • This method offers a promising approach for both basic miRNA research and clinical diagnosis of human diseases.
  • The ability to detect a large number of miRNAs from minimal sample volumes enhances its utility in biomarker discovery and diagnostics.