Method and apparatus for determining a probability of colorectal cancer in a subject

1. A method of determining whether a human test subject requires an invasive screening procedure for colon cancer, the method comprising:

identification of whether the human test subject is more likely to have colorectal cancer than to not have colorectal cancer, the identification comprising:

a) amplifying test cDNA complementary to test mRNA to obtain values of levels of test cDNA corresponding to the test mRNA expressed specifically by each gene of a set of genes consisting of IL2RB, ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 comprising the steps of:

i) generating test cDNA from the test mRNA expressed specifically by each gene of the set of genes from blood of the human test subject;

ii) reacting each test cDNA under conditions to amplify the test cDNA with a primer pair for said cDNA, wherein each primer pair consists of a forward primer and a reverse primer; and

iii) obtaining the values of the levels of the test cDNA corresponding to the test mRNA expressed specifically by each of the IL2RB, ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, and VNN1 genes:

b) obtaining a set of 6 ratios, each ratio being obtained by normalizing the level of the test cDNA corresponding to the test mRNA expressed specifically by each of the ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, and VNN1 genes against the level of the test cDNA corresponding to the test mRNA expressed by the IL2RB gene; and

c) applying each of the 6 ratios to a mathematical model formulated by analyzing, using logistic regression, a training set of training set ratios, said training set ratios being obtained from levels of cDNA corresponding to mRNA expressed by each of the ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, and VNN1 genes, in blood of training subjects having colorectal cancer and training subjects not having colorectal cancer, normalized to levels cDNA corresponding to mRNA expressed by the IL2RB gene in the blood of said training subjects, to determine the probability that a test subject has colorectal cancer as opposed to not having colorectal cancer

wherein when the human test subject is identified as more likely to have colorectal cancer than to not have colorectal cancer as a result of said identification, determining that the human test subject requires the invasive screening procedure for colon cancer, the invasive screening procedure being selected from the group consisting of colonoscopy, sigmoidoscopy, and combinations thereof.

2. The method of claim 1 , wherein the forward primer and reverse primer pair for ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, and VNN1 test cDNA consists, respectively, 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.

3. The method of claim 1 , wherein the conditions to amplify the test cDNA comprise:

a. combining each test cDNA with a primer pair, 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 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 primer and the second primer to hybridize with their complementary sequences on the first strand and the second strand of the test cDNA, and to allow the thermostable DNA polymerase to extend the primers; and

d. repeating steps (b) and (c).