Detection of lung neoplasia by amplification of RNA sequences
Inventors
Allawi, Hatim • Lidgard, Graham P. • Krueger, Chateen • Kaiser, Michael W. • Sander, Tamara J.
Assignees
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Publication Number
US-12098431-B2
Publication Date
2024-09-24
Expiration Date
Abstract
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.
Core Innovation
The invention describes a method of preparing a set of amplified DNAs by extracting RNA from a sample of blood, serum, plasma, or saliva from a human subject and using reverse transcription to form a reference cDNA from at least one reference RNA and marker cDNA from up to eight marker RNAs. The marker RNAs are selected from GAGE12D, FAM83A, LRG1, MAGEA4, XAGE-1 d, SFTPB, AKAP4, and CYP24A1.
The reverse transcribing and amplifying occur in one or more reaction mixtures comprising primer oligonucleotides for the reference RNA and the marker RNAs, reverse transcriptase, and thermostable DNA polymerase. Marker cDNA and reference cDNA are subsequently used to amplify up to eight marker DNAs and reference DNA.
In the described application context, lung cancer/neoplasia screening is performed using an RT-QuARTS single-tube RT-PCR configuration that combines invasive flap endonuclease cleavage and FRET cassette fluorescence readout. Multiplex mRNA markers including GAGE12D, FAM83A, LRG1, XAGE-1 d, MAGEA4, SFTPB, AKAP4, and CYP24A1 are assayed with normalization to a reference RNA such as CASC3, β-actin mRNA, U1 snRNA, or U6 snRNA, generating diagnostic records without opening the reaction vessel for real-time fluorescence detection.
Claims Coverage
The document provides one independent method claim and its main claim coverage focuses on preparing amplified DNA sets from human RNA samples using reverse transcription for reference and multiple marker RNAs (up to eight), followed by separate amplification of marker DNAs and reference DNA in reaction mixtures containing primer oligonucleotides, reverse transcriptase, and thermostable DNA polymerase. The inventive features span the selection of specific marker RNAs and reference RNAs, primer design defined by SEQ ID NOs, and the combined RT and amplification reaction mixture composition.
Human sample RNA for reference and marker cDNA
Providing RNA extracted from a sample of blood, serum, plasma, or saliva from a human subject; and 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.
Multiplex marker RNA selection for up to eight markers
Selecting the up to eight marker RNAs from the group consisting of GAGE12D, FAM83A, LRG1, MAGEA4, XAGE-1 d, SFTPB, AKAP4, and CYP24A1 marker RNAs.
Reference and marker amplification from corresponding cDNAs
Amplifying up to eight marker DNAs from the marker cDNA; and amplifying reference DNA from the reference cDNA.
Primer oligonucleotides with SEQ ID-defined primer targeting
In the reverse transcribing and amplifying, comprising 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 for each marker from a first marker-specific primer and a second marker-specific primer complementary to a sequence corresponding to the listed SEQ ID NOs or its complement.
Reaction mixture composition with reverse transcriptase and thermostable DNA polymerase
The one or more reaction mixtures comprising reverse transcriptase and thermostable DNA polymerase.
Overall, the claims cover a workflow that converts RNA from human blood, serum, plasma, or saliva into reference cDNA and multiplex marker cDNA (up to eight specified markers), then amplifies marker DNAs and reference DNA using primer oligonucleotides with SEQ ID-defined target sequences, in reaction mixtures that include reverse transcriptase and thermostable DNA polymerase.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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