Method to measure gene expression ratio of key genes
OwnerPierre Aman, Anders Stalberg +1inventors
InventorsPierre Aman, Anders Stalberg, Mikael Kubista
Life of this patent
Estimating the term…
Abstract
The invention is a method to determine the amounts, in particular the relative amounts, of nucleic acids in complex biological samples by means of real-time PCR. According to the invention the biological sample is systematically diluted and each dilution is studied by real-time PCR for all genes of interest. From the dependence of the threshold cycle on dilution factor for each of the genes, the PCR efficiencies of the reactions are determined in the particular samples. Determining also the relative sensitivity of the real-time PCR assays compared, the relative amounts of two nucleic acids in complex biological samples are determined with unprecedented accuracy.
Claims11
1-20. (canceled)
A method for diagnosing and/or classifying a disease by comparing the expression ratio of two genes by determining the ratio of the corresponding mRNAs in a sample comprising the steps:
- a. converting the mRNAs to cDNA and;
- b. performing one or more controlled dilutions of the sample and;
- c. amplifying each of the two genes in the sample, and in each said dilutions of the sample by PCR amplification while registering the number of amplification cycles required to obtain a preset signal threshold level (CT) and;
- d. estimating the efficiency (E) of the PCR for each of the two genes in the particular sample from the dependence of CT on the logarithm of the dilution factor whereby the relative amounts of two genes present in a sample is determined using a relation:
- N
- 0
-
- A
- N
- 0
-
- B
- =
- K
- RS
-
-
- (
- 1
- +
- E
- A
- )
- CT
- A
- (
- 1
- +
- E
- B
- )
- CT
- B
- wherein N0 reflects the number of units, N, present at the time zero of cDNA of the two genes, and the relative sensitivity KRS reflects a difference in the fluorescence and binding efficiencies of primers used in the two PCR assays.
Dependencies are inferred from the claim text.