RNA isolation from soluble urine fractions
View Patent ↗The invention provides methods for isolating RNA from the soluble fraction of urine. The methods can be used for detecting the presence or absence of an RNA, or quantifying the amount of an RNA. The methods are useful for diagnosing an individual suspected of having a disease by detecting the level of RNA associated with the disease in the soluble fraction of urine. The methods are also useful for prognosing an individual diagnosed with a disease by detecting the level of RNA associated with the disease in the soluble fraction of urine.
1. A method for processing urine for detection of RNA expression associated with a benign prostate hyperplasia, comprising:
a) separating urine sediment from the soluble urine fraction of a urine sample obtained from an individual suspected of having cancer, wherein RNA associated with the benign prostate hyperplasia is present in the soluble urine fraction; and
b) concentrating the soluble urine fraction by ultrafiltration to produce a soluble urine concentrate, wherein the volume of the soluble urine concentrate is reduced at least 50% from the original urine volume,
wherein the benign prostate hyperplasia-associated RNA is selected from the group consisting of HSPD1, IMPDH2, PDLIM5, and UAP1.
2. The method of claim 1 , wherein urine sediment is separated from the soluble urine fraction by centrifugation or filtration.
3. The method of claim 1 , wherein said ultrafiltration comprises a filter having a nominal molecular weight limit of not more than about 100,000 daltons.
4. The method of claim 1 , wherein said ultrafiltration comprises a filter having a nominal molecular weight limit of not more than about 50,000 daltons.
5. The method of claim 1 , wherein said ultrafiltration comprises a filter having a nominal molecular weight limit of between about 3,000 daltons and about 10,000 daltons.
6. The method of claim 1 , wherein said ultrafiltration comprises a filter having a nominal molecular weight limit of about 3,000 daltons.
7. The method of claim 1 , wherein the RNA is isolated from the soluble urine concentrate by solid phase extraction.
8. The method of claim 1 , wherein said urine sediment is removed by centrifugation or filtration.
9. A method for amplifying nucleic acids from a urine sample for detection of RNA expression associated with a benign prostate hyperplasia, comprising;
a) separating urine sediment from the soluble urine fraction of a urine sample obtained from an individual suspected of having cancer, wherein RNA associated with the benign prostate hyperplasia is present in the soluble urine fraction,
b) concentrating the soluble urine fraction by ultrafiltration to produce a soluble urine concentrate, wherein the volume of the soluble urine concentrate is reduced at least 50% from the original urine volume,
c) isolating RNA from the soluble urine concentrate produced in step (b),
d) reverse transcribing the RNA from the soluble urine concentrate produced in step (c) to form cDNA; and
e) amplifying the cDNA produced in step (d),
wherein the benign prostate hyperplasia-associated RNA is selected from the group consisting of HSPD1, IMPDH2, PDLIM5, and UAP1.
10. The method of claim 9 , wherein urine sediment is separated from the soluble urine fraction by centrifugation or filtration.
11. The method of claim 9 , wherein said ultrafiltration comprises a filter having a nominal molecular weight limit of not more than about 50,000 daltons.
12. The method of claim 9 , wherein said ultrafiltration comprises a filter having a nominal molecular weight limit of between about 3,000 daltons and about 10,000 daltons.
13. The method of claim 9 , wherein said ultrafiltration comprises a filter having a nominal molecular weight limit of about 3,000 daltons.
14. The method of claim 9 , wherein the RNA is isolated from the soluble urine concentrate by solid phase extraction.
15. The method of claim 9 , wherein said urine sediment is separated from said soluble fraction by centrifugation or filtration.
16. The method of claim 9 , wherein the RNA is mammalian RNA.
17. A method for isolating nucleic acids from a urine sample for detection of RNA expression associated with a benign prostate hyperplasia, said method comprising,
a) providing a urine sample from an individual suspected of having a cancer,
b) processing said urine sample to produce a cell-free urine sample, wherein RNA associated with the benign prostate hyperplasia is present in the cell-free urine sample, and
c) subjecting said cell-free urine sample to ultrafiltration using a filter having a nominal molecular weight limit of between about 3 kDa and about 100 kDa, to produce a soluble urine concentrate, wherein the RNA associated with the cancer is retained in the soluble urine concentrate, and wherein the volume of the soluble urine concentrate is reduced at least 50% from the original urine volume,
wherein the benign prostate hyperplasia-associated RNA is selected from the group consisting of HSPD1, IMPDH2, PDLIM5, and UAP1.
18. The method of claim 17 , wherein the cell-free urine sample is produced by centrifugation or filtration.
19. The method of claim 17 , wherein said ultrafiltration comprises the use of a filter having a nominal molecular weight limit of not more than about 50,000 daltons.
20. The method of claim 17 , wherein said ultrafiltration comprises the use of a filter having a nominal molecular weight limit of between about 3,000 daltons and about 10,000 daltons.
21. The method of claim 17 , wherein said ultrafiltration comprises the use of a filter having a nominal molecular weight limit of about 3,000 daltons.
22. The method of claim 17 , further comprising isolating the nucleic acid from the soluble urine concentrate produced in step (c) using solid phase extraction.
23. The method of claim 22 , further comprising amplifying the isolated nucleic acid.
24. A method for processing urine for detection of RNA expression associated with benign prostate hyperplasia comprising concentrating a urine sample from an individual suspected of having benign prostate hyperplasia to produce a soluble urine concentrate wherein RNA associated with benign prostate hyperplasia present in the urine sample is retained in the soluble urine concentrate, wherein the volume of the soluble urine concentrate is reduced at least 50% from the original urine volume, and wherein the benign prostate hyperplasia-associated RNA is selected from the group consisting of HSPD1, IMPDH2, PDLIM5, and UAP1.