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Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction

  • US 5,876,978 A
  • Filed: 06/16/1997
  • Issued: 03/02/1999
  • Est. Priority Date: 04/06/1993
  • Status: Expired due to Term
First Claim
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1. A method for quantitative measurement of gene expression of target genes comprising:

  • a) isolating cellular mRNA of target genes and housekeeping genes which are reverse transcribed and specifically amplified in the presence of competitive templates such that a ratio of each target gene to each housekeeping gene is obtained and that ratio is used to assess the amount of gene expression of each target gene by conducting simultaneous polymerase chain reaction amplification of a mixture of the following;

    i) at least one oligonucleotide primer pair of each target gene,ii) at least one oligonucleotide primer pair of each housekeeping gene,iii) at least one mutated competitive template of each target gene,iv) at least one mutated competitive template of each housekeeping gene, andv) native cDNA which contains at least one copy of each target gene cDNA and at least one copy of each housekeeping gene cDNA;

    to form polymerase chain reaction cDNA products comprising;

    native cDNA of each target gene and each housekeeping gene, and mutated cDNA of each target gene and each housekeeping gene;

    in which an appropriate amount of a mixture of competitive templates for different target genes at known concentrations relative to one another and that contain different quantified amounts of housekeeping genes relative to target genes is included in the PCR amplification mixture such that the native and competitive templates of the target genes and housekeeping genes being assessed can both be visualized following amplification by i) preparing a sufficient master mixture containing aliquots of cDNA, competitive template mixture, dNTPs, thermostable DNA polymerase and buffer for the number of genes to be measured;

    ii) placing an aliquot of the reaction mixture in a number of reaction vessels on ice corresponding to the number of genes to be evaluated, and iii) placing an aliquot of primers specific to one of the genes to be evaluated in each reaction vessel and placing the primers for the housekeeping gene in a separate tube,(b) isolating the cDNA products;

    (c) detecting the relative presence of the native cDNA products and mutated cDNA products by comparing the amount of native cDNA coding for each target gene and the amount of mutated cDNA coding for the competitive template of target gene to the amount of native cDNA coding for each housekeeping gene and the amount of mutated cDNA coding for the competitive template of each housekeeping gene;

    wherein comparison of the relative presence of the amounts of native cDNA products and mutated cDNA products of the target gene and the housekeeping gene provide the quantitative measurement of the expression of the target gene; and

    ,(d) measuring the ratio of the density of the band corresponding to the PCR product for the native gene relative to the density of the band corresponding to the PCR product for the competitive template for each gene;

    wherein, for each gene, the native/competitive template density ratio for the target gene is divided by the native/competitive template density ratio for the housekeeping gene to provide a final value in units of mRNAs/106 housekeeping gene mRNAs.

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