Early Detection Research Network

Pancreatic Reference Set Application- Ann Killary and Subrata Sen-MD Anderson (2012)

Pancreatic Ref Set App-Killary-Sen(2012)
366
Feng, ZidingFred Hutchinson Cancer Research Center
TNC/TFPI,
No design specified.
Genomics
G.I. and Other Associated Cancers Research Group

ELISA assays. We propose to screen a two gene panel (TNC/TFPI) from functional genomic pathways approaches, by ELISA assays, in a collection of blinded plasma samples with the PCCG. In subsequent years, we will add to this panel additional candidates identified in the parent EDRN grant that are in the migration signature network or the 20q amplicon. For ELISA assays, we will utilize commercially available kits. Plasma levels of the predominant isoform of TNC, TNC-large variant (TNC-L) will be determined using a Human Tenascin-C Large (HMV) (FNIII-B) ELISA kit (IBL-America, Minneapolis, MN), which detects human TNC high molecular weight variant by sandwich ELISA. Plasma TFPI levels will be determined using a commercially available Quantikine Human TFPI ELISA kit (DTFP10) (R&D Systems, Inc. Minneapolis, MN), which detects predominantly free TFPI and a very small percentage of LDL and HDL-bound TFPI by sandwich ELISA. Additional ELISA assays will be added in years 2-3 using ingenuity network analysis to identify secreted proteins that interact with migration signature proteins identified using functional genomic approaches. In addition, Dr. Sen’s lab has identified candidate biomarkers mapping with the 20q amplicon and which are both amplified and overexpressed as well as are secreted proteins. In years 2-3, we will examine all candidate markers by ELISA to determine 20q pathway markers increase the sensitivity and specificity of the 3p panel. Validation of a 4 miR Panel. For assaying the levels of miRs in plasma, qRT-PCR assays will be performed, as described (9). For the selected panel of miRs, we propose to develop a high throughput qRT-PCR assay by adapting our published method to a 96 well format where the critical steps of purification, such as LiCl precipitation and DNAse treatments will be done in successive steps in the same plates. In years 2-3, additional miRs that target both the 20q amplicon genes as well as miRs that target migration genes in Dr. Killary’

ELISA assays. We propose to screen a two gene panel (TNC/TFPI) from functional genomic pathways approaches, by ELISA assays, in a collection of blinded plasma samples with the PCCG. In subsequent years, we will add to this panel additional candidates identified in the parent EDRN grant that are in the migration signature network or the 20q amplicon. For ELISA assays, we will utilize commercially available kits. Plasma levels of the predominant isoform of TNC, TNC-large variant (TNC-L) will be determined using a Human Tenascin-C Large (HMV) (FNIII-B) ELISA kit (IBL-America, Minneapolis, MN), which detects human TNC high molecular weight variant by sandwich ELISA. Plasma TFPI levels will be determined using a commercially available Quantikine Human TFPI ELISA kit (DTFP10) (R&D Systems, Inc. Minneapolis, MN), which detects predominantly free TFPI and a very small percentage of LDL and HDL-bound TFPI by sandwich ELISA. Additional ELISA assays will be added in years 2-3 using ingenuity network analysis to identify secreted proteins that interact with migration signature proteins identified using functional genomic approaches. In addition, Dr. Sen’s lab has identified candidate biomarkers mapping with the 20q amplicon and which are both amplified and overexpressed as well as are secreted proteins. In years 2-3, we will examine all candidate markers by ELISA to determine 20q pathway markers increase the sensitivity and specificity of the 3p panel. Validation of a 4 miR Panel. For assaying the levels of miRs in plasma, qRT-PCR assays will be performed, as described (9). For the selected panel of miRs, we propose to develop a high throughput qRT-PCR assay by adapting our published method to a 96 well format where the critical steps of purification, such as LiCl precipitation and DNAse treatments will be done in successive steps in the same plates. In years 2-3, additional miRs that target both the 20q amplicon genes as well as miRs that target migration genes in Dr. Killary’
Elisa

There are currently no biomarkers annotated for this protocol.

No datasets are currently associated with this protocol.