PMC:4795482 / 15637-19943 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4795482","sourcedb":"PMC","sourceid":"4795482","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4795482","text":"2.2. qPCR (Experiment 2)\nThese same subjects in Experiment 1 were used for qPCR, plus ~90 additional subjects for Experiment 2 (shown in Supplementary Table 1). Total RNA was extracted from five brain regions (dorsolateral prefrontal cortex (DLPFC), amygdala, hippocampus, nucleus accumbens, and cerebellum) for each subject using the method outlined in Section 2.1. Total RNA from the DLPFC, amygdala, hippocampus, nucleus accumbens, and cerebellum were used for making complementary DNA (cDNA) (Table 2). cDNA was generated using TaqMan reverse-transcription (RT) reagents according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA), and cDNAs were aliquoted and stored at −20 °C. In brief, the cDNA synthesis contained 5 µL of 10× Taqman RT buffer; 11 µL of 25 mM MgCl2; 10 µL of deoxy NTPs; 2.5 µL of Oligo d(T)16 primer; 1 µL of RNase inhibitor; 1.25 µL of Multiscribe reverse transcriptase, and 1 µL of RNA (1 µg/µL). Two separate 50 µL reactions for each RNA were performed and combined together. Each cDNA batch reaction had a maximum of 24 tubes to ensure the best sample quality. The primers were designed using Primer Express software (Applied Biosystems) and purchased from Bioneer, Inc. Factors including melting temperature and guanine-cytosine (GC) content were considered. The HLA-DPA1 forward and reverse primers were designed to hybridize to sequences located in exon 3, near the site of hybridization for the probe (Probe Set 2950343; Affymetrix, Inc.). The primer set was BLAST searched against the entire human genomic sequence database for specificity, and primers used are shown in Supplementary Methods, Part 3 for all genes. The HLA-DPA1 primers were tested by using a set of cDNAs from cerebellum), genomic DNA, no template control (NTC), and RT minus (two individual DLPFC RNAs without cDNA). The primer test results showed that all cDNA amplified with a single band, while the NTC and gDNA amplified greater than 40. The RT minus showed greater than six cycles difference from the cDNA samples. This detection ensured that the HLA-DPA1 primers were specific to HLA-DPA1. Similar procedures were used for HLA-DRB1 and CD74 qPCR analyses.\nQuantitative PCR (qPCR) was performed on an ABI 7900HT Sequence Detection System (Applied Biosystems) in 384-well plates. The samples were aliquoted by the Biomek3000 Robot (Beckman Coulter, Brea, CA, USA) and run in triplicate using one plate per gene. The reaction was performed in a 12.5 µL total volume with 6.25 µL of 2× SYBR Green Master Mix (Applied Biosystems); 0.25 µL of 10 µM forward primer; 0.25 µL of 10 µM reverse primer; 2 µL of a 1:10 dilution of cDNA template (corresponding to approximately 4 ng RNA), and water to a total volume of 12.5 µL. The thermal cycle conditions were: 50 °C for 2 min (incubation), 95 °C for 10 min (activation), 45 cycles at 95 °C for 15 s (denaturation), and 60 °C for 1 min (annealing/extension), and a final dissociation step at 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s. The qPCR cycle threshold (Ct) was set in the middle of the exponential phase of the amplification. In each experiment, the individual sample was run in triplicate and the Ct of each well was recorded at the end of the reaction. The mean and standard deviation (SD) of the three Cts were calculated and the average value was accepted if the triplicate Ct values were within ±1 Ct. Two representative qPCR runs of HLA-DPA1 robotically pipetted in 384-well assay plates were examined for two brain regions, and three wells were eliminated. The average coefficient of variation was ~0.8% for each plate. The relative quantification was used to measure gene expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase complex subunit A (SDHA) were selected as the housekeeping genes. After correction with the mean of the two housekeeping genes (Ct target − Ct mean of housekeeping), an ANCOVA was used for the average delta Ct values for each subject. A repeated-subjects ANCOVA was used with factorial blocks of diagnosis, region, and SNP rs9277341, and also included age, RIN, and pH covariates. The fold change in gene expression was calculated to elucidate the direction of differences in mRNA levels between diagnosis and control samples in each brain region.","divisions":[{"label":"Title","span":{"begin":0,"end":24}}],"tracks":[]}