Method for quantitative measurement of gene expression for indentifying individuals at risk for bronchogenic carcinoma

Abstract

A method measure expression of multiple target genes in a progenitor cell for bronchogenic carcinoma comprising the use of reverse transcription-polymerase chain reaction (RT-PCR) to allow simultaneous expression measurement of the multiple target genes is disclosed.

I claim:  
     
         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 −14 M 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.