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Universal stem-loop primer method for screening and quantification of microRNA.
Li-hong Yang1, Si-lu Wang2, Li-li Tang2
1Clinical Laboratory Diagnostic Centre, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
Plos One
|December 31, 2014
Summary
A new universal stem-loop primer (USLP) method simplifies microRNA (miR) quantification. This cost-effective approach offers rapid, precise, and sensitive screening of miR profiles, significantly reducing time and expense compared to traditional methods.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Quantitative microRNA (miR) expression analysis is crucial in biological research.
- Traditional stem-loop RT-PCR (TSLP) for miR quantification is time-consuming, reagent-intensive, and inconvenient.
- A need exists for a more efficient and cost-effective method for miR profiling.
Purpose of the Study:
- To develop and validate a novel 'universal stem-loop primer' (USLP) method for microRNA (miR) screening and semi-quantification.
- To assess the efficiency, precision, sensitivity, and cost-effectiveness of the USLP method compared to traditional stem-loop primer (TSLP) methods.
- To screen miR profiles in phytohemagglutinin (PHA)-stimulated T cells using the USLP method.
Main Methods:
- Development of a universal stem-loop primer (USLP) with 8 random nucleotides at the 3' end.
- Application of USLP for screening 87 candidate miRs in PHA-stimulated human peripheral blood T cells.
- Validation using dissociation curve analysis, gel electrophoresis, and assessment of precision (intra-class correlation coefficient - ICC) and sensitivity.
Main Results:
- The USLP method demonstrated specificity comparable to TSLP.
- Significant changes in miR expression were observed: miR-150-5p decreased ~10-fold and miR-155-5p increased ~7-fold post-PHA treatment.
- USLP exhibited high precision (ICC < 2.5%), sensitivity up to 10^3 copies/µl, and reduced primer costs by 75% and test time by 60% compared to TSLP.
Conclusions:
- The universal stem-loop primer (USLP) method is a simple, rapid, precise, and sensitive approach for microRNA (miR) profiling.
- USLP offers significant cost and time savings compared to traditional stem-loop RT-PCR (TSLP).
- This innovative method is well-suited for high-throughput screening of miR expression profiles.
