title: SRAN Human 270 v1.0
status: Public on Apr 22 2016
contact.city: Springfield
contact.first: Sophia
contact.last: Ran
contact.state: IL
contact.address: 801 N. Rutledge St.
contact.department: MMICB
contact.country: USA
contact.institute: Southern Illinois University - School of Medicine
organism: Homo sapiens
description: RT-qPCR assay
  GoTaq Green Master Mix (Promega, WI)
manufacture_protocol: In-house human inflammatory and lymphatic-specific RT-qPCR targeted array was constructed from known and putative inflammatory and lymphatic genes. The array contains a total of 269 targets, including 67 cytokines, 57 cytokine receptors, 18 chemokines, 11 chemokines receptors, 10 known TLR4 downstream targets, 64 other inflammation-related proteins, 12 genes expressed primarily in lymphatic endothelial cells, and 32 endothelial cell associated proteins. An accession number based on CDS was identified for each gene of interest on the NCBI database (Bethesda, MD). Accession numbers were used to obtain potential primer sequences via the NIH qPrimerDepot (http://primerdepot.nci.nih.gov/) or Harvard Primerbank (http://pga.mgh.harvard.edu/primerbank/index.html). The sequence of each primer was compared against all known nucleotide sequences in the human genome using the NCBI BLAST alignment tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Only primers that had unique specificity to the individual target gene were used for validation. Primer DNA oligos were ordered from Integrated DNA Technology (Coralville, IA). Each primer was validated using four dilutions (1, 1:10, 1:100, and 1:1000) of human universal cDNA tested in duplicate with a final primer concentration of 0.5µM. RT-qPCR was performed with GoTaq Green Master Mix (Promega, WI) on a MasterCycle realplex PCR machine (Eppendorf, NY). The quality of each primer was confirmed by melting curve analysis and the amplification efficiency by Ct slope analysis of the four cDNA dilutions. All primer sequences are listed with the normalized Ct values.