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Related Concept Videos

MicroRNAs01:22

MicroRNAs

4.2K
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
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MicroRNAs01:22

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MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After...
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A Robust Protocol to Quantify Circulating Cancer Biomarker MicroRNAs.

Emma Bell1,2, Hannah L Watson1, Shivani Bailey1

  • 1Department of Pathology, University of Cambridge, Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.

Methods in Molecular Biology (Clifton, N.J.)
|April 26, 2017
PubMed
Summary

This study presents an optimized protocol for quantifying microRNAs (miRNAs) in serum using multiplexing techniques. The method enhances sensitivity and efficiency for circulating miRNA detection, even with limited sample input.

Keywords:
Cerebrospinal fluidMicroRNAPlasmaPre-amplificationRT-qPCRSerum

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genomics

Background:

  • Reverse-transcriptase quantitative PCR (RT-qPCR) is standard for microRNA (miRNA) quantification in cells and tissues.
  • Quantifying circulating miRNAs in serum poses unique analytical challenges.
  • Existing methods may require substantial sample volumes or lack sensitivity.

Purpose of the Study:

  • To develop an optimized protocol for sensitive and efficient quantification of circulating miRNAs.
  • To enable miRNA profiling from small serum sample volumes.
  • To establish a reliable method for assessing serum sample quality and miRNA levels.

Main Methods:

  • Utilized multiplexing of Taqman miRNA stem-loop primers during reverse transcription.
  • Incorporated a multiplexed pre-amplification step to boost assay sensitivity.
  • Employed standard Taqman qPCR methodology for final miRNA quantification.

Main Results:

  • Successfully quantified a panel of up to 20 miRNAs from limited serum input.
  • Demonstrated enhanced sensitivity for detecting low-abundance circulating miRNAs.
  • Validated a protocol for assessing serum sample quality and miRNA expression.

Conclusions:

  • The optimized protocol offers a sensitive and efficient approach for circulating miRNA analysis.
  • This method facilitates miRNA biomarker discovery from small serum samples.
  • The protocol is suitable for high-throughput miRNA profiling in clinical and research settings.