Claims
- 1. A method to measure expression of multiple target genes in a progenitor cell for bronchogenic carcinoma comprising:
using reverse transcription-polymerase chain reaction (RT-PCR) to allow simultaneous expression measurement of the multiple target genes.
- 2. The method of claim 1, which quantitative competitive RT-PCR is used to measure mRNA levels of glutathione-S-transferases (GSTs) and glutathione peroxidases (GSHPxs) in the progenitor cell.
- 3. The method of claim 2, in which the progenitor cell comprises a bronchial epithelial cell.
- 4. The method of claim 3, in which at least one of the mRNA levels of the following glutathione-S-transferases are measured: mGST, GSTM3, combined GSTM1, 2, 4, 5, GSTT1, GSTP1, GSHPx, and GSHPxA.
- 5. The method of claim 4, in which the levels of GSTP1, GSTM3 and GSHPx are significantly lower in normal bronchial epithelial cell than in bronchogenic carcinoma cells.
- 6. The method of claim 4, in which a gene expression index is evaluated by multiplying the values for: mGST×GSTM3×GSHPx×GSHPxA×GSTP1.
- 7. The method of claim 4, in which a gene expression index is evaluated by multiplying the values for: mGST×GSTM3×GSHPx×GSTP1.
- 8. The method of claim 4, in which a gene expression index is evaluated by multiplying the values for: GSTP1×mGST×GSHPx.
- 9. The method of claim 4, in which a gene expression index is evaluated by multiplying the values for: GSTP1×GSHPx×GSTM3.
- 10. The method of claim 4, in which a gene expression index is evaluated by multiplying the values for: mGST×GSTM3×GSHPx.
- 11. The method of claim 4, in which a gene expression index is evaluated by multiplying the values for: GSTM3×GSHPx.
- 12. The method of claim 4, in which a gene expression index is evaluated by multiplying the values for: GSTM×GSTP1×mGST.
- 13. The method of claim 6, in which sensitivity for detecting normal bronchial epithelial cells as compared to bronchogenic carcinoma cells is about 90%.
- 14. The method of claim 6, in which specificity for detecting normal bronchial epithelial cells as compared to bronchogenic carcinoma cells is about 76%.
- 15. The method of claim 1, comprising
a) coamplifying a housekeeping gene along with the target genes (to control for the amount of cDNA included in the reaction); b) including known amounts of cDNA competitive templates (CTs) for both the target genes and the housekeeping gene; c) identifying, choosing primers for synthesizing the competitive templates (CTs) and for amplification of native template (NT) and CT sequences; d) comparing the levels of the housekeeping gene CTs to the target gene CTs where the ratio of housekeeping gene CT to each of the target gene CTs is the same; e) preparing a master mix that contains the components: dNTPs, buffer, water, Taq polymerase, cDNA and aliquot of CT solution containing known concentrations of CTs for the housekeeping gene and the target genes; f) specifying each gene to be amplified in each reaction by the primers included in each reaction by aliquoting separately from the master mix; g) determining the amount of cDNA loaded for each sample by comparing the density of PCR product band for housekeeping gene NTcDNA to PCR product band for housekeeping gene CTcDNA; and h) determining quantitative expression of the target genes.
- 16. The method of claim 15, in which the quantitative expression of the target genes is determined by:
a) calculating a ratio of target gene NT to CT product; and b) dividing the calculated number of target gene NT molecules by the calculated number of housekeeping gene NT molecules to correct for loading differences.
- 17. The method of claim 15, in which the housekeeping gene comprises β-actin.
- 18. The method of claim 17, in which the concentration of the competitive templates (CTs) in each PCR reaction is 10−14M for β-actin and varied for each of the other genes.
- 19. A method for determining a patient who is at risk for developing cancer by assessing peripheral blood lymphocyte DNA for polymorphesisms in a regulatory region of target genes that are associated with high or low expression of the target genes.
- 20. The method of claim 19, in which quantitative competitive RT-PCR is used to measure mRNA levels of glutathione-S-transferase (GSTs) and glutathione peroxidases (GSHPxs) in a progenitor cell.
Government Interests
[0001] The present invention was made under a Research Grant No. NIH-P01 ES07168 from the National Institute of Health who may have certain rights thereto. The present invention relates generally to a method for the quantitative measurement of gene expression using multiplex competitive reverse transcription polymerase chain reaction (MC RT-PCR). To identify individuals at risk for bronchogenic carcinoma.
PCT Information
Filing Document |
Filing Date |
Country |
Kind |
PCT/US02/07259 |
3/12/2002 |
WO |
|
Provisional Applications (1)
|
Number |
Date |
Country |
|
60275854 |
Mar 2001 |
US |