Method of predicting chemotherapeutic responsiveness of cancer
Disclosed is a method of predicting clinical tumor outcome by providing gene expression from a tumor sample. The method utilizes a novel genetic screen to identify genes that contribute to chemotherapeutic responsiveness, using formalin fixed paraffin embedded clinical samples of epithelial cancer, specifically serous ovarian cancer. The method is useful in predicting tumor responsiveness to chemotherapeutics, including alkylating agents, cisplatin, antimetabolites, plant alkaloids, and antitumor antibiotics. A microarray screen showed formalin fixed paraffin embedded samples can identify genes related to chemotherapeutic response with 86% efficiency.
1. A method of measuring DNA expression of genes in a formalin-fixed paraffin-embedded clinical sample, where the genes are provided in FIG. 1 , comprising the steps of:
extracting a biological material from the formalin-fixed paraffin-embedded clinical sample, wherein the biological material is RNA, further comprising:
subjecting the formalin-fixed paraffin-embedded clinical sample to xylene;
lysing cells in the sample with a detergent and a proteinase inhibitor solution;
generating cDNA from the RNA, further comprising:
fragmenting the RNA;
subjecting the fragmented RNA to reverse transcriptase Polymerase Chain Reaction to form cDNA; and
obtaining a plurality of nucleic acid expressions in the clinical sample further comprising:
contacting the cDNA with a probeset to form the plurality of nucleic acid expressions, wherein the probeset is provided in FIG. 1 ;
measuring the hybridization of the cDNA to the probeset to provide a measure of DNA expression of genes in a formalin-fixed paraffin-embedded clinical sample.
2. The method of claim 1 , wherein the RNA is extracted from the formalin-fixed paraffin-embedded clinical sample by the steps of:
precipitating the RNA using ethanol after the cells are lysed with the detergent and the proteinase inhibitor solution; and
collecting the precipitated RNA;
wherein the detergent and the proteinase inhibitor solution is made of sodium dodecyl sulfate and Proteinase K.
3. The method of claim 1 , wherein the formalin-fixed paraffin-embedded clinical sample is cut into 5-10 μm slices prior to subjecting the formalin-fixed paraffin-embedded clinical sample to xylene.
4. The method of claim 1 , further comprising treating the formalin-fixed paraffin-embedded clinical sample subjected to xylene with ethanol to form an ethanol-treated formalin-fixed paraffin-embedded clinical sample.
5. The method of claim 4 , further comprising centrifuging the ethanol-treated formalin-fixed paraffin-embedded clinical sample.
6. The method of claim 5 , wherein the ethanol-treated formalin-fixed paraffin-embedded clinical sample is centrifuged at 12,000×g.
7. The method of claim 1 , wherein the detergent and proteinase inhibitor solution is 10% sodium dodecyl sulfate/Proteinase K buffer.
8. A method of measuring DNA expression in a formalin-fixed paraffin-embedded clinical sample, where the genes for the DNA expression are provided in FIG. 1 , comprising the steps of:
extracting a biological material from a formalin-fixed paraffin-embedded clinical sample, wherein the biological material is RNA, further comprising:
subjecting the formalin-fixed paraffin-embedded clinical sample to xylene;
lysing cells in the sample with a 10% SDS/Proteinase K buffer solution;
generating cDNA from the RNA, further comprising:
fragmenting the RNA;
subjecting the fragmented RNA to reverse transcriptase Polymerase Chain Reaction to form cDNA; and
obtaining a plurality of nucleic acid expressions in the clinical sample further comprising subjecting the biological material to a probeset, wherein the probeset is provided in FIG. 1 .
9. The method of claim 8 , wherein the RNA is extracted from the formalin-fixed paraffin-embedded clinical sample by the steps of:
precipitating the RNA using ethanol after the cells are lysed with the detergent and the proteinase inhibitor solution; and
collecting the precipitated RNA.
10. The method of claim 8 , wherein the formalin-fixed paraffin-embedded clinical sample is cut into 5-10 μm slices prior to subjecting the formalin-fixed paraffin-embedded clinical sample to xylene.
11. The method of claim 8 , further comprising treating the formalin-fixed paraffin-embedded clinical sample subjected to xylene with ethanol to form an ethanol-treated formalin-fixed paraffin-embedded clinical sample.
12. The method of claim 11 , further comprising centrifuging the ethanol-treated formalin-fixed paraffin-embedded clinical sample.
13. The method of claim 12 , wherein the ethanol-treated formalin-fixed paraffin-embedded clinical sample is centrifuged at 12,000×g.