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

• US 5,643,765 A
• Filed: 04/06/1993
• Issued: 07/01/1997
• Est. Priority Date: 04/06/1993
• Status: Expired due to Term

• Current Assignee: University of Rochester
• Original Assignee: University of Rochester
• Inventors: Willey, James C.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/043,390
• Time in Patent Office: 1,547 Days
• Field of Search: 435/91.2, 435/91.51, 435/6, 536/24.33
• 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 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 for quantitative measurement of gene expression through multiplex competitive reverse transcriptase polymerase chain reaction amplification has been established which comprises isolating cellular mRNA of a target gene and a housekeeping gene which are then reverse transcribed and specifically amplified in the presence of competitive templates such that a ratio of target to housekeeping gene is obtained and used to assess the amount of gene expression. This method is useful for analysis of the small specimens (cells and tissues) available from human subjects and will allow improved understanding of mechanisms of disease.

Claims

1. Method for quantitative measurement of expression of a target gene comprising:

a) isolating at least a portion of a RNA sequence which encodes the RNA for the target gene and a portion of an RNA for a housekeeping gene, which RNA has been isolated from a sample cell;

b) subjecting the RNA sequence of step a) and two internal mutated standards comprising single base routants of the target gene and the housekeeping gene cDNA that cause either a loss or gain of an EcoRI restriction endonuclease recognition site to reverse transcription to obtain at least one native cDNA which encodes the DNA for both the target gene and at least one native cDNA which encodes the DNA for the housekeeping gene;

c) conducting polymerase chain reaction amplification of the native cDNA in the presence of target gene and housekeeping gene primers and a predetermined quantified amount of the routate internal standards;

d) conducting a quantification step of products of the polymerase chain reaction amplification of step c) by

i) maximizing heterodimer formation,

ii) calculating the quantities of the products, and

iii) comparing samples of the products simultaneously using a master mixture which is the same for each product and having the same dilution of internal standards;

e) subjecting the amplified cDNA of step c) to enzyme restriction by digesting with EcoRI;

f) subjecting the digested cDNA of step e) to electrophoresis to separate wild type from mutated products; and

f) measuring the expression of the target gene relative to the housekeeping gene to provide the quantitative measurement of expression of the target gene.

2. The method of claim 1, wherein the housekeeping primer comprises at least one oligonucleotide homologous to the cDNA of a housekeeping gene selected from the group comprising GAPDH, β

-actin, 28S RNA and 18S RNA genes and containing known restriction endonuclease recognition site sequences or one or two base pair mismatches for restriction endonuclease recognition sequences.

3. The method of claim 1, wherein the housekeeping primer comprises at least two oligonucleotides that contain sequences homologous to sequences in the housekeeping genes, which housekeeping primer sequences are useful for generating artificially shortened competitive templates.

4. The method of claim 1, in which the quantification step is conducted at a non-exponential phase of the polymerase chain reaction amplification.

5. The method of claim 1, in which the quantification step comprises calculating the quantities of the products as an odds ratio of native target gene/mutated target gene to native housekeeping gene/mutated housekeeping gene.

6. The method of claim 1, in which the master mixture comprises approximately 1×

PCR buffer (50.0 mM KCl, 10.0 mM Tris-HCl, 1.5 mM MgCl₂), 25 pmoles of primers coding for the target gene and the housekeeping gene, 0.2 mM dNTP'"'"'s (A,T,C,G) and constant amounts of both internal standards per 100 μ

L reaction mixture.

7. A method for quantitative measurement of gene expression of a target gene comprising:

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

i) at least one oligonucleotide primer pair of at least one target gene,

ii) at least one oligonucleotide primer pair of at least one housekeeping gene,

iii) at least one mutated competitive template of the target gene,

iv) at least one mutated competitive template of the housekeeping gene, and

v) native cDNA which contains at least one copy of the target gene cDNA and at least one copy of the housekeeping gene cDNA;

to form polymerase chain reaction cDNA products comprising;

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

(b) isolating the cDNA products; and

,

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

8. The method of claim 7, 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.

9. The method of claim 8 in which the point mutation comprises one or two base pair mismatch.

10. The method of claim 9 in which the point mutation causes either a gain or a loss of an EcoRI restriction endonuclease recognition site.

11. The method of claim 7 in which the target gene and the housekeeping gene have the same recognition site.

12. The method of claim 7 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.

13. The method of claim 12 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.

14. The method of claim 13 in which the cDNA products are digested with EcoRI.

15. The method of claim 7 in which the mutated competitive templates of the target gene and the housekeeping gene comprise artificially shortened competitive templates.

16. The method of claim 7 in which the polymerase chain reaction amplification is allowed to proceed to a point which maximizes heterodimer formation.

17. The method of claim 7 in which at least one housekeeping gene is selected from the group comprising glyceraldehyde phosphate dehydrogenase (GAPDH), β

-actin or 28S RNA.

18. The method of claim 7 in which the mutated internal standard competitive templates are prepared by site directed mutagenesis.

19. The method of claim 7 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.

20. A method for quantitative measurement of gene expression of a target gene in tissue samples comprising:

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

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

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

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

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

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 the target gene oligonucleotide primers, the housekeeping gene oligonucleotide primers, and predetermined quantities of the housekeeping gene competitive template and the target gene competitive template,

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.

21. The method of claim 20 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.

22. The method of claim 20 in which polymerase chain reaction products are denatured at about 94°

C. An slowly cooled to allow heterodimer formation.