Ultrasensitive quantification of multiplexed mRNA variants via splice-junction anchored DNA probes and SplintR ligase-initiated PCR

Abstract

A method based on mRNA-templated ligation of splice-junction anchored DNA probes followed by PCR amplification of the ligated product has been developed for multiplexed detection of mRNA splice variants with...

Specific recognition and sensitive quantification of mRNA splice variants via one-pot ligation-dependent loop-mediated isothermal amplification

“…[37][38][39] SYBR Green I, a dsDNA-specific fluorescent intercalator, was employed to perform the real-time measurement in a label-free and cost-effective manner. More importantly, different from our previously developed ligationdependent RNA sensing strategies, [25][26][27][28][29][30][31][32] the ligation reaction and LAMP are sequentially carried out in one tube, which greatly simplifies the experimental process for mRNA alternative splicing variant analysis and meets the routine measurements of ordinary laboratories.…”

“…4,25 Taking the high specificity of ligases and the exponential nucleic acid replication features of different amplification technologies, many pioneering ligation-mediated methods have been developed for precise analysis of mRNA alternative splicing processes and accurate quantification of splicing variants as well as other RNA targets. 25–32 These ligation-based strategies not only simplify the primer/probe design but also significantly improve the specificity of these methods. However, series open-tube experimental procedures may cause disastrous cross-contamination, leading to false-positive signals, greatly challenging the reliability and accuracy of quantification results.…”

“…Third, the two stem-loop DNA probes for different splicing isoforms are identical except for the anti-target sequences and different target-dependent LAMP can be performed with the universal primers, which can simplify the probe/primer design and reduce the amplification bias. More importantly, compared to the previously developed ligation-based sensing strategies, [25][26][27][28][29][30][31][32] the ligation reaction and LAMP are sequentially carried out and monitored in one tube, which can greatly simplify the experimental process and effectively avoid cross-contamination and false-positive results arising from multiple open-tube operations and testing, 33 which can greatly promote the reliability and accuracy of the proposed one-pot ligation-LAMP.…”

Specific multiplexed detection of mRNA splice variants based on size-coding DNA probes and universal PCR amplification

“…30,31 Ligation-dependent PCR assay can specifically quantify mRNA variants by the ligation of splicejunction anchored probes. 32,33 Despite many attempts, specific, multiple, and highly sensitive in vitro detection assays are still lacking for routine diagnostic analysis of RNA splice variants.…”

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection

“…Usually, DNA amplification is a must for sensitive NAT. Although various principles have been proposed for isothermal amplification, PCR is the most reliable, simple, and effective way for DNA amplification, especially for multiplex detection; thus it has been commonly used as a gold standard for pathogen detection. , However, PCR needs complicated instrumentation for both amplification and detection, greatly limiting the application in POCT, which requires amplification and detection in a simple and portable format …”