Sensitive and specific miRNA detection method using SplintR Ligase

Abstract

We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR® Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4–6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection.

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

“…Based on published estimates, SplintR mediated direct RNA detection outperforms randomhexamer-RT FISSEQ for targeted transcriptomics by 2-3 orders of magnitude in detection efficiency (~0.005% vs ~20%), and circumvents random-primed RT related artifact and over-10 | P a g e representation of abundant housekeeping RNA like rRNA [3][4][5]19,21 . Further, this approach retains the key advantages of the barcoded padlock probe system (multiplexing and targeting sensitivity), without the need for RT or cost-prohibitively expensive LNA modified primers 6,8 .…”

Evaluation of miRNA Detection Methods for the Analytical Characteristic Necessary for Clinical Utilization

“…Contrary to this, especially in world of miRNA molecules, this issue does not pay much attention and necessary analytical characteristics of miRNA detection methods are not being tested. Therefore, we evaluated three miRNA detection technologies that are based on different principles: well-known RT-qPCR, RT step is based on using specific (TaqMan miRNA assay) or universal (TaqMan ADVANCED assay) priming strategy; miREIA technology, using hybridization and specific antibody to DNA/RNA hybrids [19]; SplintR-qPCR, a combination between previously mentioned methods, which utilizes hybridization and ligation step followed by qPCR [18]. …”

“…SplintR-qPCR, a combination between previously mentioned methods, which utilizes hybridization and ligation step followed by qPCR [18]. …”

“…The SplintR-qPCR procedure was obtained from Jin et al [18] and modified in-house. The hybridization mix was composed of: 1 μl of probe A (1 pmol/μl); 1 μl of probe B (1 pmol/μl), 2 μl of hybridization buffer (1% triton X-100, 10% BSA, 50% tris-buffered saline), 0.02 μl of RNAse inhibitor (Sigma Aldrich) and 4.98 μl of nuclease-free water.…”

“…Multiple miRNAs have already been determined by SplintR-qPCR using a double-quenched TaqMan probe. Only a 4–6 bp overlap between a DNA probe and miRNA was required for efficient ligation, providing more flexibility in probe design and allowing it to reach higher specificity [18]. …”