# US 8,568,971 B2 · Methods and compositions involving microRNA

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- Number: US 8,568,971 B2
- Document ID: US8568971B2
- Status: Granted
- Kind: B2
- Application number: 12/890,398
- Filed: 2010-09-24
- Published: 2011-05-12
- Granted: 2013-10-29
- Earliest claimed: 2004-05-28
- Assignees: Asuragen, Inc.
- Applicants: David Brown; Rick Conrad; Eric Devroe; Marianna Goldrick; Kerri Keiger; Emmanuel Labourier; Ivonne Moon; Patricia Powers; Jeffrey Shelton; Jaclyn Shingara
- Inventors: David Brown; Rick Conrad; Eric Devroe; Marianna Goldrick; Kerri Keiger; Emmanuel Labourier; Ivonne Moon; Patricia Powers; Jeffrey Shelton; Jaclyn Shingara
- Web page: https://patentgrep.com/patent/US8568971B2

## CPC

- A61P 35/00: Antineoplastic agents
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/3517: Marker; Tag
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2320/10: in screening processes
- C12Q 2600/158: Expression markers
- C12Q 2600/178: miRNA, siRNA or ncRNA
- Y02A 90/10: Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention concerns methods and compositions for isolating, enriching, and/or labeling miRNA molecules and for preparing and using arrays or other detection techniques for miRNA analysis. Moreover, the present invention concerns methods and compositions for generating miRNA profiles and employing such profiles for therapeutic, diagnostic, and prognostic applications.

## Claims

### Claim 1

A method for identifying a human patient suspected of having cancer as having a marker correlated with cancer based on an increase in miR-21 expression comprising:
a) analyzing miR-21 expression in a biological sample from the patient comprising cells suspected of being cancerous; and,
b) identifying the patient as i) having a marker correlated with cancer if an increase in miR-21 expression compared to a noncancerous biological sample is detected in the biological sample or ii) as not having a marker correlated with cancer if the increase fails to be detected, wherein the compared biological samples are derived from the same tissue type.

### Claim 2 (depends on 1)

The method of claim 1, wherein the biological sample from the patient comprises breast tissue and the increase in miR-21 expression is correlated with breast cancer.

### Claim 3 (depends on 2)

The method of claim 2, further comprising analyzing the sample for 1) an increase in miR-200B and/or 2) a decrease in miR-100, miR-126, miR-143, miR-145, and/or miR-205 compared to a noncancerous breast sample.

### Claim 4 (depends on 1)

The method of claim 1, wherein the biological sample from the patient comprises colon tissue and the increase in miR-21 expression is correlated with colon cancer.

### Claim 5 (depends on 4)

The method of claim 4, further comprising analyzing the sample for 1) an increase in miR-31, miR-106A, miR-200B, miR-203, or miR-223 and/or 2) a decrease in miR-125a, mir-130a, miR-133, miR-135A, miR-143, or miR-145 compared to a noncancerous colon sample.

### Claim 6 (depends on 1)

The method of claim 1, wherein the biological sample from the patient comprises prostate tissue and the increase in miR-21 expression is correlated with prostate cancer.

### Claim 7 (depends on 6)

The method of claim 6, further comprising analyzing the sample for 1) an increase in miR-15A, miR-29, or miR-141 and/or 2) a decrease in miR-188, miR-290, or miR-331 compared to a noncancerous prostate sample.

### Claim 8 (depends on 1)

The method of claim 1, wherein the biological sample from the patient comprises thyroid tissue and the increase in miR-21 expression is correlated with thyroid cancer.

### Claim 9 (depends on 8)

The method of claim 8, further comprising analyzing the sample for 1) an increase in miR-15A or miR-200B and/or 2) a decrease in miR-30A-as, miR-31, miR-135A, miR-138, miR-152, miR-199A-as, miR-203, or miR-331 compared to a noncancerous thyroid sample.

### Claim 10 (depends on 1)

The method of claim 1, wherein the biological sample from the patient comprises bladder tissue and the increase in miR-21 expression is correlated with bladder cancer.

### Claim 11 (depends on 10)

The method of claim 10, further comprising analyzing the sample for 1) an increase in miR-9 as, miR-182, or miR-200B and/or 2) a decrease in miR-133, miR-143, or miR-145 compared to a noncancerous bladder sample.

### Claim 12

A method for identifying a human patient as having a marker correlated with cancer based on an increase in miR-21 expression comprising:
a) obtaining from a patient a breast, colon, lung, thyroid, or bladder tissue sample comprising cells suspected of being cancerous; and,
b) identifying the patient, based on a determination of the tissue sample, as i) having a marker correlated with cancer if there is an increase in miR-21 expression in the tissue sample compared to a noncancerous tissue of the same type as the tissue sample or ii) as not having a marker correlated with cancer if the increase fails to be detected.

### Claim 13 (depends on 11)

The method of claim 11, wherein the tissue sample comprises breast cells.

### Claim 14 (depends on 11)

The method of claim 11, further comprising treating the patient for cancer after the patient is identified as having a marker correlated with cancer.

### Claim 15 (depends on 11)

The method of claim 11, wherein the tissue sample comprises breast cells and the increase in miR-21 expression is correlated with breast cancer.

### Claim 16 (depends on 15)

The method of claim 15, further comprising analyzing the sample for 1) an increase in miR-15a, miR-16, miR-24, miR-25, or miR-200B and/or 2) a decrease in miR-99, miR-100, miR-126, miR-135A, miR-143, miR-145, miR-197, miR-205, miR-211, miR-298 compared to a noncancerous breast sample.

### Claim 17 (depends on 11)

The method of claim 11, wherein the tissue sample comprises colon tissue and the increase in miR-21 expression is correlated with colon cancer.

### Claim 18 (depends on 17)

The method of claim 17, further comprising analyzing the sample for 1) an increase in miR-31, miR-106A, miR-200B, miR-203, or miR-223 and/or 2) a decrease in miR-125a, mir-130a, miR-133, miR-135A, miR-143, or miR-145 compared to a noncancerous colon sample.

### Claim 19 (depends on 11)

The method of claim 11, wherein the tissue sample comprises thyroid tissue and the increase in miR-21 expression is correlated with thyroid cancer.

### Claim 20 (depends on 19)

The method of claim 19, further comprising analyzing the sample for 1) an increase in miR-15A or miR-200B and/or 2) a decrease in miR-30A-as, miR-31, miR-135A, miR-138, miR-152, miR-199A-as, miR-203, or miR-331 compared to a noncancerous thyroid sample.

### Claim 21 (depends on 11)

The method of claim 11, wherein the tissue sample comprises bladder tissue and the increase in miR-21 expression is correlated with bladder cancer.

### Claim 22 (depends on 21)

The method of claim 21, further comprising analyzing the sample for 1) an increase in miR-9 as, miR-182, or miR-200B and/or 2) a decrease in miR-133, miR-143, or miR-145 compared to a noncancerous bladder sample.

### Claim 23

A method for identifying a patient as having a marker correlated with cancer based on an increase in miR-21 expression comprising:
a) analyzing miR-21 expression in a breast, colon, lung, thyroid, or bladder sample from a patient suspected of having cancer; and,
b) identifying the patient as having a marker correlated with cancer if an increase in miR-21 expression of at least 50% in the patient's biological sample compared to a noncancerous comparable sample is determined; or identifying the patient as not having a marker correlated with cancer if the increase is not determined.

### Claim 24 (depends on 23)

The method of claim 23, further comprising analyzing the sample for a decrease in miR-143 and/or an increase in miR-200b compared to a noncancerous comparable sample.

### Claim 25

A method for identifying a patient in need of cancer treatment comprising
a) analyzing miR-21 expression in a biological sample from the patient comprising breast cells suspected of being cancerous;
b) detecting an increase in miR-21 expression compared to a noncancerous breast cell sample; and,
c) identifying the patient as having a marker correlated with breast cancer.

### Claim 26 (depends on 25)

The method of claim 25, further comprising analyzing the sample for 1) an increase in miR-200B and/or 2) a decrease in miR-100, miR-126, miR-143, miR-145, and/or miR-205 compared to a noncancerous breast sample.

### Claim 27

A method for identifying a human patient as having a marker correlated with breast cancer based on an increase in miR-21 expression comprising:
a) obtaining from a patient a breast tissue sample comprising cells suspected of being cancerous; and,
b) identifying the patient, based on a determination of the tissue sample, as i) having a marker correlated with breast cancer if there is an increase in miR-21 expression in the tissue sample compared to a noncancerous tissue of the same type as the tissue sample or ii) as not having a marker correlated with cancer if the increase fails to be detected.

### Claim 28 (depends on 12)

The method of claim 12, wherein the patient has an increase in miR-21 expression and is identifying as having a marker correlated with breast cancer.