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) providing RNA extracted from a sample of blood, serum, plasma, or saliva from a human subject;
b) from the RNA, reverse transcribing to form a reference cDNA from at least one reference RNA and to form marker cDNA from up to eight marker RNAs, wherein the up to eight marker RNAs are selected from the group consisting of GAGE12D, FAM83A, LRG1, MAGEA4, XAGE-1 d, SFTPB, AKAP4, and CYP24A1 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 LRG1-specific primer hybridized to LRG1 RNA within a sequence corresponding to SEQ ID NO:33,
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,
XAGE-1 d marker RNA is reverse transcribed using a first XAGE-1 d-specific primer hybridized to XAGE-1 d RNA within a sequence corresponding to SEQ ID NO:36,
SFTPB marker RNA is reverse transcribed using a first SFTPB-specific primer hybridized to SFTPB RNA within a sequence corresponding to SEQ ID NO:35,
AKAP4 marker RNA is reverse transcribed using a first AKAP4-specific primer hybridized to AKAP4 RNA within a sequence corresponding to SEQ ID NO:28, and
CYP24A 1 marker RNA is reverse transcribed using a first CYP24A1-specific primer hybridized to CYP24A1 RNA within a sequence corresponding to SEQ ID NO:30;
c) amplifying up to eight marker DNAs from the marker cDNA; and
d) amplifying reference DNA from the reference cDNA,
wherein the reverse transcribing and the amplifying occur in one or more reaction mixtures comprising:
i) primer oligonucleotides for reverse transcribing the reference RNA and for amplifying reference DNA from the at least one reference cDNA, and primer oligonucleotides for reverse transcribing the up to eight marker RNAs and for amplifying the up to eight marker DNAs from the marker cDNA, wherein the primer oligonucleotides comprise up to eight primer pairs selected from:
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,
the first MAGEA4-specific primer and a second MAGEA4-specific primer complementary to SEQ ID NO:34 or its complement,
the first XAGE-1 d-specific primer and a second XAGE-1 d-specific primer complementary to SEQ ID NO:36 or its complement,
the first SFTPB-specific primer and a second SFTPB-specific primer complementary to SEQ ID NO:35 or its complement,
the first AKAP4-specific primer and a second AKAP4-specific primer complementary to SEQ ID NO:28 or its complement, and
the first CYP24A1-specific primer and a second CYP24A1-specific primer complementary to SEQ ID NO:30 or its complement,
ii) reverse transcriptase; and
iii) thermostable DNA polymerase.
2. The method of claim 1 , wherein reverse transcribing RNAs and amplifying DNAs from cDNAs occur in a single reaction mixture.
3. The method of claim 1 , wherein amounts of marker and reference DNAs amplified from 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 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 each of the up to eight marker DNAs amplified from the marker cDNA are all measured in separate reaction mixtures.
11. The method of claim 10 , wherein an amount of a marker DNA amplified from the marker cDNA and an 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 comprises one or more marker RNAs selected from GAGE12D, FAM83A, LRG1, and MAGEA4 marker RNAs.
13. The method of claim 1 , wherein the up to eight marker RNAs comprises one or more marker RNAs selected from XAGE-1 d, SFTPB, AKAP4, and CYP24A1 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 cell-free sample.
16. The method of claim 1 , wherein the reverse transcriptase is MMLV reverse transcriptase.