Claims
- 1. A method for quantitative measurement of gene expression of target genes comprising:
deparaffinizing a formalin-fixed paraffin-embedded biological tissue sample; rehydrating the deparaffinized sample; combining the rehydrated sample with a chaotropic solution having a pH of about 5 and comprising guanidinium isothiocyanate and a reducing agent; homogenizing and heating the sample and chaotropic solution mixture at a temperature of about 95° C. for about 30 minutes; recovering the sample's RNA by extraction from the chaotropic solution using a water insoluble organic solvent; purifying the recovered RNA; converting the purified RNA to cDNA by a reverse transcription reaction; subjecting the cDNA to a PCR reaction in a polymerase chain reaction solution that comprises an oligonucleotide probe suitable for amplifying at least a specified sequence, a polymerase and a fluorochrome; measuring the change that occurs in the intensity of fluorescence as a result of the PCR reaction; and determining, on the basis of the change in the intensity of the fluorescence, the quantity of a nucleic acid having a specified sequence present in the sample.
- 2. The method of claim 1wherein the concentration of guanidinium isothiocyanate in the chaotropic solution is about 3M.
- 3. The method of claim 2 wherein
the reducing agent comprises dithiothreitol.
- 4. The method of claim 3 wherein
the water insoluble organic solvent comprises chloroform.
- 5. The method of claim 4 wherein
the recovered RNA is purified using ethanol precipitation.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser. No. 09/797,216, filed on Mar. 1, 2001, now allowed, which is a divisional of U.S. application Ser. No. 09/469,338, filed on Dec. 20, 1999, now U.S. Pat. No. 6,248,535, which are hereby incorporated by reference in their entirety for all purposes as if fully set forth herein.
GOVERNMENT SUPPORT
[0002] The government has certain rights in this invention pursuant to grant number R01 CA 71716 from the National Cancer Institute of the National Institutes of Health.
Divisions (1)
|
Number |
Date |
Country |
Parent |
09469338 |
Dec 1999 |
US |
Child |
09797216 |
Mar 2001 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09797216 |
Mar 2001 |
US |
Child |
10631413 |
Jul 2003 |
US |