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Universal TaqMan-Based Detection System for RNA Quantification

Published:
Lead Inventor: Tong-Chuan He
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SUMMARY

  • Reverse transcriptase quantitative PCR (qPCR) effectively quantifies coding and noncoding RNA expressions in cells and tissue samples. Among various RT-qPCR detection systems, SYBR Green-based system is most commonly used, although it often lacks desirable specificity; fluorescent probe-based qPCR (e.g., TaqMan) offers high sensitivity and specificity, but its utility is limited by high costs of probe synthesis.
  • The current technology is a universal TaqMan-based detection system, that allows cost-effective analysis of coding and noncoding transcriptomics, with broad applications in research and clinical diagnostics.
  • The inventor designed five degenerate hairpin primers that contain the same hairpin and universal TaqMan recognizing sequences, and randomized nucleotides at the 3’-end. These primers underwent four rounds of linear regression-based machine learning analysis to identify the optimal DHP mix (COD24). COD24 replicated transcript-specific hairpin primers in mRNA quantification and removes the necessity of using transcript-specific primers. The COD24 system was further validated in quantifying lncRNAs and miRNAs with high sensitivity and specificity.
  • Using universal TaqMan-based probes can drastically reduce the cost associated with probe synthesis.The current technology should be readily adapted for other forms of fluorophore probe-based qPCR detection chemistry. 

FIGURE

COD24-based RT-qPCR expression quantification of coding and non-coding RNA with high specificity. Quantification of RNA expression (A). Total RNA was isolated from 143B (a), Mel-624 (b), Mel-888 (c), A375 (d), SJSA1 (e), and UC-MSC (f) cells, and subjected to RT reactions using the TSP and COD24 primers, followed by TaqMan qPCR analysis of the 8 selected genes. Quantification of lncRNA expression (B) and miRNA (C). Total RNA was isolated from HEK-293 cells, and subjected to RT reactions with COD24 primers, lncRNA-specific primers (TSPs) or miRNA-specific primers (TSPs), followed by TaqMan qPCR analysis of the selected lncRNAs (n=10) or miRNAs (n=14).

 

 

ADVANTAGES

ADVANTAGES

  • Cost Effective
  • Streamline Probe Design and Synthesis
  • Highly Sensitive and Specific
  • Detection of Coding and Noncoding RNAs

APPLICATIONS

  • Coding and Noncoding Transcriptomic Analysis
  • Clinical Diagnostics
  • Basic and Translational Research