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An optimized universal hairpin primer system for RT-qPCR-based quantification of miRNA expression

MicroRNAs (miRNAs) are single-stranded non-coding RNA molecules (containing ≈22 nucleotides) found in plants, animals, and some viruses. In addition to exploring the evolutionary history and cellular functions of miRNA, researchers are investigating the clinical significance of miRNA dysregulation. Circulating miRNAs in blood and cerebrospinal fluid also have the potential to serve as biomarkers in several diseases. Reverse transcription quantitative PCR (RT-qPCR) analysis is currently the most popular method for quantifying miRNA levels. However, there are two primary technical challenges for qPCR-based miRNA detection: the short length of miRNAs and highly conserved sequences present in miRNA families. Using stem-loop or hairpin primers for miRNA RT reactions helps to overcome these challenges (Figure 1A). However, the hairpin primer system requires using miRNA-specific hairpin primers (MsHPs), which is not cost-effective or conducive to high throughput.

In a recent Nucleic Acids Research article, He et al. reported the development of a novel universal hairpin primer (UHP) system that allows RT-qPCR-based quantification of miRNAs without requiring MsHPs. Using four UHPs that share the same hairpin structure but are anchored with two, three, four and six degenerate nucleotides at their 3’- ends (Figure 1B; UHP2, UHP3, UHP4 and UHP6), the group analyzed the accuracy of UHP- versus MsHP-based RT-qPCR. The touchdown-qPCR reactions that formed the core of their analyses were set up using Biotium’s Forget-Me-Not™ EvaGreen qPCR Master Mix. Targeting a panel of 14 miRNAs and using material from several commonly studied human cell lines, they found that the UHPs yielded robust RT products and quantified miRNAs with adequate efficiency. UHP4 proved to be the “winning” universal primer, demonstrating comparable accuracy with MsHPs. UHP4 was also shown to perform as well as MsHPs in the presence of ribosomal RNA and long transcript contamination. In further experiments, the authors empirically determined an optimized UHP (OUHP) mix that closely mirrored the abilities of MsHP-based miRNA quantification. With further development, including potential adaptation for multiplex analyses, a UHP system could overcome the necessity of using MsHPs for RT reactions and make these analyses more cost-effective and amenable to high-throughput studies.

Figure 1. Graphical representation of the UHP system. (A) An MsHP contains six nucleotides complementary to the 3′-end of mature miRNA, followed by a stem–loop structure. Once an MsHP anneals to the targeted miRNA (a), the RT reaction is carried out (b). The resultant product is then used as a template for RT-qPCR (c) using a forward primer specific to the 5′-end of the mature miRNA and a reverse primer complementary to the 3′-end of the hairpin. (B) The structure and nucleotide sequences of the tested UHPs. “MsHP” is a representative MsHP that contains a 14-bp stem, 16-nt loop and six nucleotides complementary to 3′-end of mature miRNA (indicated as ‘x’). UHP2, UHP3, UHP4 and UHP6 illustrate the four UHPs tested in this study. These share the same hairpin sequence as MsHPs, except that they contain two, three, four and six randomized nucleotides at the stem’s 3′-end. Further experiments (data not shown) identified an optimized UHP mix with the molar composition of UHP2:UHP4:UHP6 = 8:1:1 that closely recapitulated MsHPs in miRNA quantification. Credit: He et al. reproduced under the Creative Commons license.

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Full Citation

He, F., Ni, N., Wang, H., et al. OUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression. Nucleic acids research, 50(4), e22-e22 (2022).

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