The present invention provides a method of quantification of a target nucleic acid, using at least any two of the genes SYT10, EPHA3, PLEKHF1 and KBTBD4 as control genes. In particular, the combination of the genes SYT10, EPHA3, PLEKHF1 and KBTBD4, known as the 4Plex, is provided as a control for nucleic acid quantification. The 4Plex has particular utility as a control for nucleic acid quantification by methylation-specific droplet digital PCR.

A method for specifically and efficiently quantifying the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5′-Dbs-adapter to the 5′-end of target RNAs, wherein the 5′-Dbs-adapter has a stem-loop structure whose protruding 5′-end base-pairs with the 5′-end of target RNAs, and wherein the loop region of 5′-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3′Db-adapter to the 3′-end of target RNAs, wherein the 3′-Db-adapter has a stem-loop structure whose protruding 3′-end base-pairs with the 3′-end of target RNAs; ligating both adapters with target RNAs by RN12 ligation to form a “dumbbell-like” structure; and, amplifying and quantifying the ligation product by RT-PCR.

A method for specifically and efficiently quantifying the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5′-Dbs-adapter to the 5′-end of target RNAs, wherein the 5′-Dbs-adapter has a stem-loop structure whose protruding 5′-end base-pairs with the 5′-end of target RNAs, and wherein the loop region of 5′-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3′Db-adapter to the 3′-end of target RNAs, wherein the 3′-Db-adapter has a stem-loop structure whose protruding 3′-end base-pairs with the 3′-end of target RNAs; ligating both adapters with target RNAs by RN12 ligation to form a “dumbbell-like” structure; and, amplifying and quantifying the ligation product by RT-PCR.

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”).

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”).

The present invention refers to a method directed to RT-qPCR reactions, preferably performed in a one or two-step approach combining the reverse transcription and subsequent PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase with mandatory 3′.fwdarw.5′ exonuclease activity, which corrects miss-incorporated nucleotides. In particular, in this invention we present a One-step RT-PCR, preferably qPCR, method with a novel priming strategy that utilizes a novel bivalent reverse primer, wherein this primer is used for both, i) the generation of cDNA and ii) the completion of the subsequent amplification using that same cDNA as template. This bivalent reverse primer also allows end-tagging the amplicon(s) obtained so that they can be used in a variety of applications including, standard sequencing, next generation sequencing (NGS), gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing, and disease research.

The present invention refers to a method directed to RT-qPCR reactions, preferably performed in a one or two-step approach combining the reverse transcription and subsequent PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase with mandatory 3′.fwdarw.5′ exonuclease activity, which corrects miss-incorporated nucleotides. In particular, in this invention we present a One-step RT-PCR, preferably qPCR, method with a novel priming strategy that utilizes a novel bivalent reverse primer, wherein this primer is used for both, i) the generation of cDNA and ii) the completion of the subsequent amplification using that same cDNA as template. This bivalent reverse primer also allows end-tagging the amplicon(s) obtained so that they can be used in a variety of applications including, standard sequencing, next generation sequencing (NGS), gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing, and disease research.

This invention releases to systems and methods for detecting the presence and quantity of a target nucleic acid in a sample using dPCR and PIP encapsulated monodisperse droplets.

This invention releases to systems and methods for detecting the presence and quantity of a target nucleic acid in a sample using dPCR and PIP encapsulated monodisperse droplets.

Provided herein are methods and compositions for multiplex detection of a large number of actionable gene fusions with very high sensitivity and specificity. The present methods and compositions can detect ALK, RET, and ROS1 gene fusions, optionally in combination with other mutations and fusions.