title: 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.
authors.affiliation: Division of RNA Biology, New England Biolabs, Ipswich, MA 01938-2773, USA.
authors.forename: Jingmin
authors.initials: J
authors.lastname: Jin
authors.affiliation: Division of RNA Biology, New England Biolabs, Ipswich, MA 01938-2773, USA.
authors.forename: Sophie
authors.initials: S
authors.lastname: Vaud
authors.affiliation: Division of RNA Biology, New England Biolabs, Ipswich, MA 01938-2773, USA.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Zhelkovsky
authors.affiliation: Division of RNA Biology, New England Biolabs, Ipswich, MA 01938-2773, USA.
authors.forename: Janos
authors.initials: J
authors.lastname: Posfai
authors.affiliation: Division of RNA Biology, New England Biolabs, Ipswich, MA 01938-2773, USA mcreynolds@neb.com.
authors.forename: Larry A
authors.initials: LA
authors.lastname: McReynolds
mesh_terms: D000818:Animals; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization / Q000379:methods*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D015342:DNA Probes; D035683:MicroRNAs; C544354:Mirn122 microRNA, mouse; D014764:Viral Proteins; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases