The present invention relates to a digital multiplex method for detecting and/or quantifying multiple target biomolecules in a sample, said biomolecules being selected from DNA, RNA, and proteins. The present invention further relates to different applications of the digital multiplex method and to a kit.

Differentiating benign cutaneous lesions from melanoma is an imprecise and subjective endeavor. The use of micro-RNAs has been investigated, but results have not been consistent across studies and clinical applications are lacking. The invention provides new micro-RNA signatures for differentiating benign lesions from melanoma. The micro-RNA signatures are robust, being stable across detection platforms, diverse sample types, and patient populations. The diagnostic methods based on these signatures control for variations in lesion composition and sample diversity, and permit cross-platform comparisons. The micro-RNA signatures and methods are amenable to the use of samples from convenient non-invasive tape strip biopsy.

Disclosed herein are novel compositions, methods, and systems for determining whether a subject has, or is at risk of developing, or is at a given stage of a condition afflicting a tissue of interest, or determining the tissue or cell provenance of a biological sample, based on expression level of one or more of the novel miRNA and isomiR sequences disclosed herein. The compositions, methods, and systems described herein can be used to diagnose a disease or disorder, or prognose a given stage and/or progression of the disease or disorder, or determine the identity of the tissue or cell in a sample. In some embodiments, the compositions, methods, and systems described herein can be used to develop a treatment for the disease or disorder. For example, in some embodiments, the novel miRNAs can be used as therapeutics for treatment of a disease or disorder.

Disclosed are methods for detecting a target nucleic acid in a sample. The methods include contacting the sample, in the presence of a polymerase and an endonuclease, with a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of a target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.

miRNAs for in vitro diagnosis of resistance of tumors to BRAF/MEK pathway (also named as MAPK 5 pathway) inhibiting drugs and for treatment of tumors which are treated with said drugs, such as melanoma, by stimulating or inhibiting the expression of down-regulated or up-regulated miRNAs, respectively.

miRNAs for in vitro diagnosis of resistance of tumors to BRAF/MEK pathway (also named as MAPK 5 pathway) inhibiting drugs and for treatment of tumors which are treated with said drugs, such as melanoma, by stimulating or inhibiting the expression of down-regulated or up-regulated miRNAs, respectively.

The present invention includes a method for identifying an Alzheimer's disease (AD) patient prior to reaching clinical disease classification, comprising: obtaining a dataset associated with a blood, serum, or plasma sample from the patient, wherein the dataset comprises data representing the level of one or more microRNA biomarkers in the blood, serum, or plasma sample; assessing the dataset for a presence or an increase in an amount of miRNA-455-3p; determining the likelihood that the patient will develop AD patient prior to reaching clinical disease classification by detecting the presence or the increase in miRNA-455-3p to produce a score that is indicative of a likelihood of developing AD, wherein a higher score relative to a healthy control indicates that the patient is likely to have the prognosis for transitioning to classified AD, wherein the healthy control is derived from a non-AD patient with no clinical evidence of AD.

The present invention relates to the discovery that the expression levels of some RNA molecules, comprising messenger RNA (mRNA), non-coding RNA (ncRNA) and/or microRNA (miRNA), and protein can be used as a diagnostic signature to predict or monitor the bone healing ability in an acutely injured subject or in a chronic nonunion subject. In certain embodiments, the invention relates to methods and compositions useful for differentiating between a nonunion, slow healing, and/or normal healing of a fractured bone and treatment recommendations. The invention further includes a kit comprising biomarker probes for assessing the bone healing ability in an acutely injured subject or in a nonunion subject after receiving therapeutic treatment.

The present invention relates to methods for diagnosing a disease by determining via multiplex fluorescence in situ hybridization (FISH) whether or not mRNA species and/or at least one miRNA species of disease-associated biomarkers ar present in a sample obtained from a subject, and by determining by multiplex sequencing whether or not said mRNA species of disease-associated biomarkers and/or said miRNA species of disease-associated biomarkers of step (a) are present in said sample. The present invention also relates to kits for performing the methods for diagnosis as described and provided herein as well as use of such kits for performing the methods for diagnosis as described and provided herein.

This invention relates to a kit or a device for the detection of stomach cancer and a method for detecting stomach cancer, and provides a kit or a device for the detection of stomach cancer, comprising a nucleic acid(s) capable of specifically binding to a miRNA(s) in a sample from a subject, and a method for detecting stomach cancer, comprising measuring the miRNA(s) in vitro.