Method and apparatus for determining a probability of colorectal cancer in a subject
A method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer is disclosed. The method comprises, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
1. A method for identifying whether a human test subject is more likely to have colorectal cancer than to not have colorectal cancer, comprising
(a) amplifying test DNA from blood of the human test subject complementary to a test mRNA to obtain values for levels of test mRNA expressed specifically by a set of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, comprising the steps of
(i) generating test cDNA from a test RNA expressed specifically by each gene of the set of genes from blood of the human test subject; and
(ii) reacting the test cDNA under conditions to amplify DNA with a set of primer pairs consisting of: a primer pair identified as SEQ ID NO: 10 and SEQ ID NO: 11, a primer pair identified as SEQ ID NO: 19 and SEQ ID NO: 20, a primer pair identified as SEQ ID NO: 28 and SEQ ID NO: 29, a primer pair identified as SEQ ID NO: 37 and SEQ ID NO: 38, a primer pair identified as SEQ ID NO: 46 and SEQ ID NO: 47, a primer pair identified as SEQ ID NO: 55 and SEQ ID NO: 56, and a primer pair identified as SEQ ID NO: 64 and SEQ ID NO: 65, wherein the first and the second primer of each primer pair represents a pair of forward and reverse primers, to obtain the values of the levels of the test mRNAs expressed specifically by the ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 genes; and
(b) comparing the product of step (a) to the value for the level of control mRNA expressed specifically by the set of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 obtained from blood of a human control subject, the human control subject not having colorectal cancer,
wherein the value for the level of test mRNA encoded by the ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and/or VNN1 gene in the human test subject higher than the level of control mRNA encoded by the ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and/or VNN1 gene in the human control subject identifies the human test subject as more than likely to have colorectal cancer and wherein the value for the level of test mRNA encoded by the IL2RB gene in the human test subject lower than the level of control mRNA encoded by the IL2RB gene in the human control subject identifies the human test subject as more than likely to have colorectal cancer.
2. The method of claim 1 , wherein the value of the level of control mRNA expressed specifically by the set of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, is obtained by a method comprising:
(i) generating control cDNA from control RNA expressed specifically by each gene of the set of genes from blood of the human control subject; and
(ii) reacting the control cDNA under conditions to amplify DNA with a set of primer pairs consisting of: a primer pair identified as SEQ ID NO: 10 and SEQ ID NO: 11, a primer pair identified as SEQ ID NO: 19 and SEQ ID NO: 20, a primer pair identified as SEQ ID NO: 28 and SEQ ID NO: 29, a primer pair identified as SEQ ID NO: 37 and SEQ ID NO: 38, a primer pair identified as SEQ ID NO: 46 and SEQ ID NO: 47, a primer pair identified as SEQ ID NO: 55 and SEQ ID NO: 56, and a primer pair identified as SEQ ID NO: 64 and SEQ ID NO: 65, wherein the first and the second primer of each primer pair represents a pair of forward and reverse primers, to obtain the values of the levels of the control mRNAs expressed specifically by the ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 genes.
3. The method of claim 1 , wherein the conditions to amplify DNA comprise:
(A) combining the test cDNA, the set of primer pairs, a thermostable DNA polymerase, and a plurality of free nucleotides comprising adenine, thymine, cytosine and guanine in a reaction mixture
(B) heating the reaction mixture to a first predetermined temperature for a first predetermined time to separate the strands of the test cDNA from each other;
(C) cooling the reaction mixture to a second predetermined temperature for a second predetermined time under conditions to allow the first and second primers to hybridize with their complementary sequences on the first and second strands of the test cDNA, and to allow the polymerase to extend the primers; and
(D) repeating steps (B) and (C).
4. The method of claim 2 , wherein the conditions to amplify DNA comprise:
(A) combining the control cDNA, the set of primer pairs, a thermostable DNA polymerase, and a plurality of free nucleotides comprising adenine, thymine, cytosine and guanine in a reaction mixture
(B) heating the reaction mixture to a first predetermined temperature for a first predetermined time to separate the strands of the control cDNA from each other;
(C) cooling the reaction mixture to a second predetermined temperature for a second predetermined time under conditions to allow the first and second primers to hybridize with their complementary sequences on the first and second strands of the control cDNA, and to allow the polymerase to extend the primers; and
(D) repeating steps (B) and (C).