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

• Current Assignee: University of Toledo
• Original Assignee: Medical College of Ohio
• Inventors: Willey, James C., Crawford, Erin L., Weaver, David A., Jackson, Clara M., DeMuth, Jeffery P.
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/876,766
• Time in Patent Office: 624 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 937/77, 937/78
• US Class Current: 435/91.2
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/107 with a competitive internal standard/control

Abstract

A method and apparatus for quantitative measurement of gene expression through multiplex competitive reverse transcriptase polymerase chain reaction amplification are disclosed. The method and apparatus are especially useful for analysis of small specimens of cells and tissues.

Claims

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, and

v) 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.

2. The method of claim 1, in which the mutated competitive templates of the target gene and of the housekeeping gene:

a) comprise at least one oligonucleotide that contains a sequence homologous to the target gene or the housekeeping gene, respectively; and

b) contain at least one known restriction endonuclease recognition site sequence or a point mutation for the restriction endonuclease recognition sequence.

3. The method of claim 2, in which the point mutation comprises one or two base pair mismatch.

4. The method of claim 3, in which the point mutation causes either a gain or a loss of an EcoRI restriction endonuclease recognition site.

5. The method of claim 1, in which the target gene and the housekeeping gene have the same recognition site.

6. The method of claim 1, in which the cDNA products are isolated by subjecting the cDNA products to digestion with a restriction enzyme, and thereafter subjecting the digested cDNA products to electrophoresis to separate the native cDNA products from the mutated cDNA products.

7. The method of claim 6, in which the mutated competitive template of the target gene causes a loss of an EcoRI restriction site and the mutated competitive template of the housekeeping gene causes a gain of an EcoRI restriction site.

8. The method of claim 7, in which the cDNA products are digested with EcoRI.

9. The method of claim 1, in which the mutated competitive templates of the target gene and the housekeeping gene comprise artificially shortened competitive templates.

10. The method of claim 1, in which the polymerase chain reaction amplification is allowed to proceed to a point which maximizes heterodimer formation.

11. The method of claim 1, in which at least one housekeeping gene is selected from the group comprising glyceraldehyde phosphate dehydrogenase (GAPDH), β

-actin or 28S RNA.

12. The method of claim 1, in which the mutated internal standard competitive templates are prepared by site directed mutagenesis.

13. The method of claim 1, in which prior to the simultaneous polymerase chain reaction amplification for each mutated competitive template:

i) two initial polymerase chain reactions are conducted using an outer primer and an inner single base mismatched mutated internal standard competitive template primer to produce two overlapping DNA fragments;

ii) isolating and purifying the overlapping DNA fragments of the initial polymerase chain reaction of step (i) above;

iii) polymerase chain reaction amplifying each of the two overlapping DNA fragments using the outer primers only;

iv) conducting a polymerase chain reaction amplification according to claim 7 above without the primers to allow for heterodimer formation; and

v) purifying and amplifying the polymerase chain reaction products of step (iv) above and thereafter diluting to use as competitive templates.

14. A method for quantitative measurement of gene expression of different target genes in tissue samples comprising:

a) synthesizing at least one oligonucleotide primer pair of at least one housekeeping gene,

b) synthesizing at least one oligonucleotide primer pair of each target gene,

c) synthesizing at least one competitive template of each housekeeping gene,

d) synthesizing at least one competitive template of each target gene,

e) isolating at least a portion of an RNA sequence from said tissue samples,

f) subjecting the RNA sequence to reverse transcription to obtain at least one native cDNA,

g) conducting polymerase chain reaction amplification of the native cDNA in the presence of each of the target gene oligonucleotide primers, each of the housekeeping gene oligonucleotide primers, and predetermined quantities of each housekeeping gene competitive template and each target gene competitive template, 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 visualize 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,

h) subjecting amplified cDNA products of step "g" above to digestion with at least one restriction enzyme,

i) subjecting the digested cDNA to electrophoresis to separate the amplified target gene and the amplified housekeeping gene from the amplified target gene competitive template and the amplified housekeeping gene competitive template, and

j) measuring the relative expression of the target gene in at least one tissue sample by dividing a ratio of the target gene to a known amount of the competitive template of the target gene by a ratio of housekeeping gene to a known amount of the competitive template of the housekeeping gene to provide the quantitative measurement of expression of the target gene by 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.

15. The method of claim 14, which further comprises making artificially shortened competitive templates of the housekeeping gene to serve as an internal mutated standard competitive template of the housekeeping gene such that there is no need for subjecting the amplified cDNA to restriction enzyme digestion.

16. The method of claim 14, in which polymerase chain reaction products are denatured at about 94°

C. and slowly cooled to allow heterodimer formation.