Detection of lung neoplasia by amplification of RNA sequences
Provided herein is technology for lung neoplasia screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of lung cancer.
1. A method of preparing a set of amplified DNAs, the method comprising: a) extracting RNA from a sample from a human subject; b) from the extracted RNA,
i) reverse transcribing to form marker cDNAs from up to eight marker RNAs, wherein the up to eight marker RNAs comprises GAGE12D, FAM83A, LRGI, and MAGEA4 marker RNAs, wherein GAGE12D marker RNA is reverse transcribed using a first GAGE12D-specific primer hybridized to GAGE12D RNA within a sequence corresponding to SEQ ID NO:32, FAM83A marker RNA is reverse transcribed using a first FAM83A-specific primer hybridized to FAM83A RNA within a sequence corresponding to SEQ ID NO:31, LRG1 marker RNA is reverse transcribed using a first LRG/-specific primer hybridized to LRG1 RNA within a sequence corresponding to SEQ ID NO: 33 , and MAGEA4 marker RNA is reverse transcribed using a first MAGEA4-specific primer hybridized to MAGEA4 RNA within a sequence corresponding to SEQ ID NO:34;
ii) amplifying marker DNAs from the marker cDNAs; and
iii) reverse transcribing to form a reference cDNA from at least one reference RNA; and
iv) amplifying reference DNA from the reference cDNA, wherein reverse transcribing and amplifying occurs in one or more reaction mixtures comprising: primer oligonucleotides for reverse transcribing each of the up to eight marker RNAs and for amplifying DNAs from the marker cDNAs, wherein the primer oligonucleotides comprise: the first GAGE12D-specific primer and a second GAGE12D-specific primer complementary to SEQ ID NO:32 or its complement; the first FAM83A-specific primer and a second FAM83A-specific primer complementary to SEQ ID NO:31 or its complement; the first LRG1-specific primer and a second LRG1-specific primer complementary to SEQ ID NO:33 or its complement; and the first MAGEA4-specific primer and a second MAGEA4-specific primer complementary to SEQ ID NO:34 or its complement; primer oligonucleotides for reverse transcribing the reference RNA and for amplifying reference DNA from the reference cDNA; reverse transcriptase; and thermostable DNA polymerase.
2. The method of claim 1 , wherein the reverse transcribing and amplifying DNAs from marker and reference cDNAs occur in a single reaction mixture.
3. The method of claim 1 , wherein amounts of marker and reference DNA amplified from the marker and reference cDNAs are measured in real time during thermal cycling.
4. The method of claim 3 , wherein each of the one or more reaction mixtures further comprises: nucleic acid probe oligonucleotides complementary to the marker and reference DNAs amplified from the marker and reference cDNAs.
5. The method of claim 4 , wherein the nucleic acid probe oligonucleotides comprise reporter molecules.
6. The method of claim 5 , wherein the reporter molecules comprise fluorophores.
7. The method of claim 5 , wherein the reporter molecules comprise flap sequences.
8. The method of claim 7 , wherein the amounts of marker and reference DNA amplified from marker and reference cDNAs are measured in a PCR-flap assay occurring in the one or more reaction mixtures, wherein each of the one or more reaction mixtures further comprises: FEN-1 endonuclease and one or more FRET cassettes.
9. The method of claim 8 , wherein amounts of marker and reference DNA amplified from marker and reference cDNAs are all measured in a single reaction mixture.
10. The method of claim 8 , wherein amounts of marker DNA amplified from marker cDNAs are all measured in separate reaction mixtures.
11. The method of claim 10 , wherein amounts of marker DNA amplified from marker cDNAs and amount of reference DNA amplified from the at least one reference cDNA are measured in each of the separate reaction mixtures.
12. The method of claim 1 , wherein the up to eight marker RNAs is a group of four marker RNAs consisting of GAGE12D, FAM83A, LRG1, and MAGEA4 marker RNAs.
13. The method of claim 1 , wherein the up to eight marker RNAs further comprises one or more marker RNAs selected from XAGE-1 d, SETPB, AKAP4, and CYP241 marker RNAs.
14. The method of claim 1 , wherein the at least one reference RNA is selected from the group consisting of CASC3 mRNA, β-actin mRNA, U1 snRNA and U6 snRNA.
15. The method of claim 1 , wherein the sample is a tissue sample, a blood sample, a serum sample, or a sputum sample.
16. The method of claim 15 , wherein the tissue sample comprises lung tissue.
17. The method of claim 1 , wherein the reverse transcriptase is MMLV reverse transcriptase.