The stool extract was diluted to 20 μg/mL and subjected to the aptamer-based targeted proteomic screen using a library of 1129 validated aptamers (Somalogic Inc., Boulder, CO, USA), as detailed in our previous study16 . Briefly, the sample was added to aptamer-coated beads allowing for the proteins in the sample to bind to their aptamer cognates. Next, the unbound proteins were washed away and the remaining bound proteins were biotinylated. The aptamer-protein complexes were photocleaved from the original beads and then conjugated to a second streptavidin-coated bead. The proteins were then denatured allowing for the recovered aptamer oligos to be hybridized onto a custom Agilent DNA array overnight, using Agilent buffers (Agilent 5188−5221) and scanned using a microarray scanner (Agilent G4900DA). Data were extracted using Agilent Feature extraction software. Along with the stool samples, eight controls were included to allow for quality control and normalization. A “no protein” buffer blank allowed for the assessment of the background signal.
Agilent dna array
Manufactured by Agilent Technologies
The Agilent DNA array is a tool for analyzing and quantifying DNA samples. It enables researchers to study gene expression patterns and genetic variations across the entire genome. The array provides a platform for high-throughput, simultaneous analysis of thousands of DNA sequences.
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2 protocols using agilent dna array
1
Aptamer-based Targeted Proteomic Profiling of Stool
2
Plasma Proteomic Profiling Using Aptamer Assay
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Plasma samples were screened to measure the levels of 1322 distinct human proteins using an aptamer-based targeted proteomic assay, as detailed in our previous study (23 (link)). After a series of incubation steps, the recovered aptamer oligos were hybridized onto a custom Agilent DNA array overnight, using Agilent buffers (Agilent; catalog no.: 5188-5221) and scanned using a microarray scanner (Agilent; catalog no.: G4900DA) (23 (link)). Data were extracted using Agilent Feature extraction software. Along with the plasma samples, a “no protein” buffer blank allowed for the assessment of background signal. Biomarker studies and data analyses were performed at the Houston OMICs Collaborative (https://hoc.bme.uh.edu/ ), where the relative fluorescence unit values for each protein were normalized using calibrators, and hybridization normalized and median normalized protein expression values were generated. The expression values for all measured proteins are listed in supplemental Table S1 . These values were further analyzed, and statistical testing was performed using R scripts in RStudio (Posit, PBC) to evaluate the differences between sample groups.
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