miRNA fingerprint in the diagnosis of diseases
The present invention provides novel methods for diagnosing a state of health based on the determination of specific miRNAs that have altered expression levels in different conditions, e.g. disease states compared to healthy controls.
1. A method of diagnosing and/or predicting the state of health in a subject comprising the steps:
(a) providing a RNA sample from said subject, wherein said sample is a blood cell sample,
(b) providing means for determining an expression profile for a plurality of miRNA and/or other non-coding RNA molecules, wherein said means are:
(i) capture probes, wherein each capture probe comprises a sequence of at least partially complementary nucleotides to each miRNA of the plurality of miRNA or other non-coding RNA molecules, and an elongation sequence, or
(ii) miRNA-specific primers, universal reverse primers and labeled probes to conduct a polymerase chain reaction
(c1) determining the expression profile of a plurality of miRNAs and/or other noncoding RNAs in the sample of (a), by:
(i) hybridizing the miRNAs and/or other non-coding RNA molecules in the sample of (a) with the means of (b)(i), and
(ii) elongating the miRNAs and/or other non-coding RNA molecules of the sample with labeled nucleotides, and
(iii) quantifying the labeled hybridized miRNA based on the label, or
(c2) determining the expression profile of a plurality of miRNA and/or non-coding RNA molecules by:
(i) obtaining cDNA samples by an RNA reverse transcription reaction using the miRNA-specific primers of (b)(ii), and
(ii) amplifying the cDNA via polymerase chain reaction with miRNA-specific forward primers of (b)(ii), universal reverse primers of (b)(ii) and the labeled probe of (b)(ii) complementary to at least a portion of cDNA to be analyzed, and
(iii) detecting levels of miRNAs on the basis of the labeled probe,
(d) comparing a predetermined subset of miRNAs and/or other non-coding RNAs in said miRNA and/or other non-coding RNA expression profile, wherein said subset is characteristic for a particular condition to a corresponding subset of miRNAs and/or other non-coding RNAs in reference miRNA and/or non-coding RNA expression profiles obtained from a plurality of different reference subjects representing a plurality of different conditions, and
(e) calculating the probability value of said subject for the particular conditions, and
(f) optionally repeating steps (d) and (e) for at least one different particular condition, and/or
(g) optionally collecting the probability values for the particular condition to diagnose and/or predict the health state of said subject.
2. The method of claim 1 , wherein step (d) comprises:
(a) Providing a plurality of reference miRNA and/or non-coding RNA expression profiles obtained from a plurality of different reference subjects representing a plurality of different conditions,
(b) Selecting a subset of miRNAs and/or non-coding RNAs characteristic for a particular condition, and
(c) Comparing the expression of a subset of miRNAs and/or non-coding RNAs in the subject to be analyzed and in at least 2 groups of reference subject, wherein the first group suffers from a particular condition (disease) and the second group does not suffer from the particular condition.
3. The method of claim 2 , wherein the subjects of the second reference group may be healthy or may suffer from a different condition (disease).
4. The method of claim 1 , wherein the condition or disease is cancer, a cardiovascular disease, an infectious disease, an inflammatory disease or an autoimmune disease.
5. The method of claim 1 , wherein the means of step (b) are a matrix comprising a plurality of capture probes.
6. The method of claim 1 , wherein the means of step (b) are means to conduct qRT-PCR.
7. The method of claim 1 , wherein said blood cell sample is a mixture of erythrocytes, leukocytes and/or thrombocytes.
8. The method of claim 1 wherein the condition or disease is lung cancer, skin cancer, melanoma or multiple sclerosis.
9. A method of diagnosing and/or predicting the state of health in a subject comprising the steps:
(a) providing a RNA sample from said subject, wherein said sample is a blood cell sample,
(b) providing means for determining an expression profile for a plurality of miRNA and/or other non-coding RNA molecules, wherein said means are:
(i) capture probes, wherein each capture probe comprises a sequence of at least partially complementary nucleotides to each miRNA of the plurality of miRNA or other non-coding RNA molecules, and an elongation sequence, or
(ii) miRNA-specific primers, universal reverse primers and labeled probes to conduct a polymerase chain reaction
(c1) determining the expression profile of a plurality of miRNAs and/or other noncoding RNAs in the sample of (a), by:
(i) hybridizing the miRNAs and/or other non-coding RNA molecules in the sample of (a) with the means of (b)(i), and
(ii) elongating the miRNAs and/or other non-coding RNA molecules of the sample with labeled nucleotides, and
(iii) quantifying the labeled hybridized miRNA based on the label, or
(c2) determining the expression profile of a plurality of miRNA and/or non-coding RNA molecules by:
(i) obtaining cDNA samples by an RNA reverse transcription reaction using the miRNA-specific primers of (b)(ii), and
(ii) amplifying the cDNA via polymerase chain reaction with miRNA-specific forward primers of (b)(ii), universal reverse primers of (b)(ii) and the labeled probe of (b)(ii) complementary to at least a portion of cDNA to be analyzed, and
(iii) detecting levels of miRNAs on the basis of the labeled probe,
(d) providing a plurality of miRNA and/or non-coding RNA reference expression profiles obtained from a plurality of different reference subjects representing a plurality of different conditions,
(e) comparing the expression of a predetermined subset of miRNAs and/or other non-coding RNAs of the expression profile determined in step c1 or c2 to a corresponding predetermined subset of miRNAs and/or other non-coding miRNAs of the miRNA and/or non-coding RNA reference expression profiles, wherein said subset of miRNAs is characteristic for a particular condition, and
(f) calculating the probability value of said subject for the particular conditions, and
(g) optionally repeating steps (e) and (f) for at least one different particular condition, and/or
(h) optionally collecting the probability values for the particular condition to diagnose and/or predict the health state of said subject.
10. The method of claim 9 , wherein the condition or disease is cancer, a cardiovascular disease, an infectious disease, an inflammatory disease or an autoimmune disease.
11. The method of claim 9 , wherein the means of step (c1) are a matrix comprising a plurality of capture probes.
12. The method of claim 9 , wherein the means of step (c2) are means to conduct qRT-PCR.
13. The method of claim 9 , wherein said blood cell sample is a mixture of erythrocytes, leukocytes and/or thrombocytes.
14. The method of claim 9 wherein the condition or disease is lung cancer, skin cancer, melanoma or multiple sclerosis.
15. The method of claim 9 wherein the predetermined set of miRNAs is representative of melanoma.