Fluidigm biomark hd

Gene expression analysis was used to examine genes related to immune, oxidative stress, glutamate, and apoptotic pathways. Total RNA was isolated by hand from 20 mg of ipsilateral parietal cortex tissue using a RNeasy^® Mini Kit (Qiagen, Germantown, MD, USA). A total of 200 ng of yielded RNA proceeded to cDNA synthesis using Quantabio qScript XLT cDNA SuperMix (Quantabio). Multiplex qPCR was performed with Fluidigm BioMark™ HD. For each sample, 1.25 μL of the resulting cDNA was combined with 3.75 μL of Sample Pre Mix (Life Technologies TaqMan^® PreAmp Master Mix and Pooled Taqman assays) and pre-amplified for 14 cycles. The reaction products were diluted 1:5 and loaded onto the Gene expression IFC according to Fluidigm^® IFC Standard Taqman Gene expression workflow. 37 TaqMan^® gene expression assays related to immune cells, neuroinflammation, oxidative stress, apoptosis, and the glutamate pathway, and 4 housekeeping gene assays were used as detailed in Table 2. Cycle threshold (Ct) values were collected for analysis, using the 2^–ΔΔCt method.

The levels of 92 oncology related proteins were quantified in 1 ul of serum using the Proseek Oncology II Proximity Extension Immunoassay Panel (Olink, Uppsala, Sweden) as previously described [12 (link)]. Samples were randomly assigned to 96-well plates using stratified randomization based on the institution of origin, diagnosis (healthy vs. cancer), ovarian cancer subtype, age, and race (when available). Samples were run on the Proseek Oncology II panel to quantify the level of protein expression. Each sample was mixed with the Proseek Oncology II reagents according to the manufacturer’s instructions and quantified by Q-PCR using a Fluidigm^® BioMark™ HD high-throughput PCR instrument at the University of Minnesota Genomics Center. The Proseek platform includes three “interplate controls” for data normalization between plates and three “negative controls” to establish background levels. Internal controls for incubation and extension are included by Olink in each assay for quality control. The Proseek^® assay reports relative quantification on a log2 scale, as Normalized Protein eXpression (NPX) values, which was normalized according to the manufacturer’s protocols. Samples that did not pass Olink quality control were not included in the analysis.

We extracted total RNA from mouse hippocampus with the RNeasy Mini Kit (catalog no. 74104; QIAGEN) and prepared cDNA with the high-capacity RNA-to-cDNA kit (cat# 4388950; Applied Biosystems) following the manufacturer’s instructions. cDNA was further purified using QIAquick PCR purification kit following the manufacturer’s instructions (cat# 28104; QIAGEN). Gene expression analysis was performed using microarray in collaboration with the Genome Technology Access Core at Washington University. Using TaqMan probes, the relative gene expression was quantitatively measured using Fluidigm Biomark HD with integrated fluidic circuits. The data were normalized by evaluation of geometricCt mean of housekeeping genes (GAPDH, RN18S1 and ACTB) for each sample. Then the expression levels were calculated according to the ΔΔCt method.

At 11 days post injury, rats were anesthetized and decapitated. The ipsilateral hippocampus was dissected, rapidly frozen, and stored at −80°C. Gene expression analysis was used to examine genes related to vascular health, neuroinflammation, and hypoxia. Total RNA was isolated from 20 mg tissue by a RNeasy® Mini Kit (Qiagen) running by QIAcube (Qiagen). 200 ng yielded RNA were proceeded to cDNA synthesis using Quantabio qScript XLT cDNA SuperMix (Quantabio). Multiplex qPCR was performed with Fluidigm BioMark HD™. For each sample, 1.25 μl of the resulting cDNA were combined with 3.75 μl of Sample Pre Mix (Life Technologies TaqMan® PreAmp Master Mix and Pooled Taqman assays) and preamplified for 14 cycles. The reaction products were diluted 1: 5, and loaded onto the Gene expression IFC according to Fluidigm® IFC Standard Taqman Gene expression workflow. 42 TaqMan® gene expression assays related to neuroinflammation, hypoxia injury and vascular health, and 4 housekeep gene assays were used and detailed in Table 2. Cycle threshold (Ct) values were collected for analysis, using the 2^−ΔΔCt method.

Colonic samples (n = 5 per treatment group, 15 total) were collected within 10 min of euthanasia. Colonic samples (1 to 2 pellets/tube) were collected into Eppendorf tubes. All samples were immediately placed in liquid nitrogen and then stored in the laboratory at −80 °C prior to analysis.
Microbial DNA was extracted from colonic samples using the DNeasy PowerLyzer PowerSoil Kit by QIAGEN. The protocol uses a bead-beating method to extract the DNA. DNA extraction was acquired by following the manufacturer’s instructions. DNA concentration was measured using NanoDrop-1000 spectrophotometer. The full-length 16S region was amplified using a Fluidigm Biomark HD and sequenced on a Pacific Biosciences Sequel IIe at the Functional Genomics and DNA Services units at the Roy J. Carver Biotechnology Center at the University of Illinois.

Good markers obtained in screening of the G. barbadense SNPs were used to genotype 118 F2 (3-79×TM-1) individuals, two parents (G. hirsutum line TM-1 and G. barbadense line 3-79), and F1 (3-79×TM-1) as controls. KASP assays were run using the Fluidigm system in 96.96 dynamic array format, utilizing multiple arrays, and read using the Fluidigm BioMark HD (Fluidigm, San Francisco, CA). Clustering for genotyping was performed using Fluidigm SNP Genotyping Analysis software. Genotypes of the 118 F2s for successfully genotyped markers were imported into JoinMap 4.1 (Van Ooijen 2011 ), and identical markers were removed (Table S5) and linkage mapped using the maximum likelihood algorithm and Haldane’s mapping function with default parameters. Linkage groups were determined using LOD scores of 5.0 or more. Cytogenetic stocks including F1 hypo-aneuploids, each deficient for a known G. hirsutum chromosome, were also genotyped for the same markers.