Detection of Naturally occurring abortive transcripts by Base-Stacking Hybridization Assisted Ligation and PCR amplification

Abstract

Abortive transcripts (ATs) refer to nascent 2-10 nucleotides (nt) RNAs released by RNA polymerases before synthesizing productive RNAs. The quantitative detection of ATs is important for studying transcription initiation and the biological function of ATs; however, no method is available for the qualitative and quantitative assessment of such ultra-short oligonucleotides (typically shorter than 11 nt) in vivo at present, even with the LNA probes, the detection limit can only reach 11 nt. Here, we demonstrated the base stacking hybridization assisted ligation (BSHAL) technique, combined with TaqMan-MGB qPCR, can detect 4-10 nt ATs with a specificity of nucleotide resolution and a sensitivity of approximately 10 pM. By this technique, we detected endogenous ATs in cell lines, mice plasmas, and mice liver tissues, respectively, and proved that naturally occurring ATs do exist. We found that the 8 nt ATs of HMSB and Gapdh could be used as reference ATs for data normalization in Homo and mouse respectively, and 8 nt ATs of Afp and Gpc3 were suitable for use as plasma biomarkers of Hepatocellular carcinoma in mouse, indicate ATs are promising biomarkers. This study offers opportunities to study ATs and other ultra-short oligonucleotides in biological samples.

Keywords: Abortive transcripts; Base stacking hybridization; Biomarkers; Ligation; Oligonucleotides; PCR.