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

• US 5,639,606 A
• Filed: 01/28/1994
• Issued: 06/17/1997
• 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): REES, DIANE
• Application Number:
  US08/188,434
• Time in Patent Office: 1,236 Days
• Field of Search: 435/6, 435/69.1, 435/91.1, 435/91.2, 435/810, 436/501, 436/63, 536/22.1, 536/23.1, 536/24.1, 536/24.3-24.33, 935/77, 935/78, 935/88
• US Class Current: 435/6.12
• 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 is established. This method is especially useful for analysis of small specimens of cells and tissues.

Claims

1. A kit for quantitatively measuring gene expression of a target gene of interest comprising at least one pair of primers for amplification of the target gene;

at least one pair of primers for amplification of a housekeeping gene;

at least one competitive template of the target gene; and

at least one competitive template of the housekeeping gene wherein the competitive templates of the target gene and the housekeeping gene differ from the target gene and the housekeeping gene, respectively, by at least a point mutation.

2. The kit of claim 1, wherein the primers of the housekeeping gene comprise the sequences of Sequence I.D. No. 5 and Sequence I.D. No. 6.

3. The kit of claim 1, wherein the competitive template of the housekeeping gene comprises the sequences of Sequence I.D. No.5, Sequence I.D. No. 6, Sequence I.D. No. 7 and Sequence I.D. No. 8.

4. The kit of claim 1, wherein the oligonucleotide primers of the housekeeping gene comprise the sequences of Sequence I.D. No. 13 and Sequence I.D. No. 15.

5. The kit of claim 1, wherein the primers of the housekeeping gene comprise the sequences of Sequence I.D. No. 13 and Sequence I.D. No. 15.

6. The kit of claim 1, wherein the primers of the target gene comprise the sequences of Sequence I.D. No. 16 and Sequence I.D. No. 18.