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Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach
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Detection of Naturally occurring abortive transcripts by Base-Stacking Hybridization Assisted Ligation and PCR

Shaowei Qin1, Haizhu Wu1, Cailin Li1

  • 1School of Food and Health, Guilin Tourism University, Guilin, 541006, China.

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New technology detects ultra-short abortive transcripts (ATs) in vivo. This breakthrough enables the study of ATs and identifies potential biomarkers for diseases like cancer.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Abortive transcripts (ATs) are short RNA fragments (2-10 nucleotides) released during transcription initiation.
  • Current methods lack the sensitivity and specificity to detect and quantify ultra-short ATs in vivo.
  • Understanding ATs is crucial for studying transcription regulation and their biological roles.

Purpose of the Study:

  • To develop a novel technique for the qualitative and quantitative detection of ultra-short ATs in vivo.
  • To validate the presence and biological relevance of endogenous ATs in various biological samples.
  • To identify potential AT biomarkers for disease diagnosis.

Main Methods:

  • Development of the base stacking hybridization assisted ligation (BSHAL) technique.
  • Integration of BSHAL with TaqMan-MGB quantitative PCR (qPCR) for enhanced sensitivity and specificity.
  • Application of the technique to detect ATs in cell lines, mouse plasma, and mouse liver tissues.

Main Results:

  • The BSHAL-qPCR method successfully detected 4-10 nucleotide ATs with nucleotide resolution and picomolar sensitivity.
  • Endogenous ATs were detected in cell lines, mouse plasma, and liver tissues, confirming their natural occurrence.
  • Identified specific 8-nucleotide ATs (HMSB, Gapdh) as suitable references for normalization and others (Afp, Gpc3) as potential plasma biomarkers for hepatocellular carcinoma in mice.

Conclusions:

  • The BSHAL-qPCR technique provides a powerful tool for studying ultra-short oligonucleotides, including ATs, in biological systems.
  • Naturally occurring ATs exist in vivo and can be quantified with high precision.
  • ATs show promise as novel biomarkers for disease detection and monitoring.