Stem-loop RT-qPCR system for multiplex miRNA profiling and its application in wound healing-specific biomarker...

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• MicroRNAs (miRNAs) are crucial biomarkers, but their low expression levels hinder clinical applications.
• Current miRNA detection methods are costly and inefficient for large-scale studies due to separate transcription and quantification steps.

Purpose of the Study:

• To develop a strategy for designing and screening miRNA-specific stem-loop reverse transcription (RT) primers.
• To establish multiplex RT-PCR and dual-quantitative PCR (qPCR) systems for simultaneous miRNA detection.

Main Methods:

• Designed and screened miRNA-specific stem-loop RT primers for simultaneous transcription of multiple miRNAs and U6.
• Developed multiplex RT-PCR and dual-qPCR systems using TaqMan probes with different dyes for concurrent detection.
• Validated the system using cell culture samples with miR-7a-5p mimic, inhibitor, or negative control.

Main Results:

• Successfully established multiplex RT-PCR and dual-qPCR systems for 21 differentially expressed miRNAs during wound healing.
• The developed system accurately quantified cell culture samples.
• Demonstrated efficient design, screening, and analysis of stem-loop RT primers for multiplex miRNA detection.

Conclusions:

• The proposed strategy enables efficient multiplex miRNA detection, offering higher automation, accuracy, and lower costs compared to commercial kits.
• This approach has practical value for clinical diagnosis and large-scale miRNA research.