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 APPLICATION DATA
[0001] This application is a divisional of U.S. patent application Ser. No. 09/469,338 filed Dec. 20, 1999.
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 |