Abi prism 7900ht sequence detection

Quantitative real-time PCR was performed on the ABI PRISM 7900HT sequence detection system using Power SYBR Green 1-Step kit (Applied Biosystems) following the manufacturer’s instructions. A minimum of three biological replicates were performed for each sample and a tomato TIP41 gene was used as internal standard^{57 (link)}. TIP41 and gene-specific primers are listed in Supplemental Table 5.

Total RNA was isolated from iPSCs using TRIzol reagent (Thermo Fisher Scientific), and quantitative real-time reverse transcription (RT)-PCR was performed using a QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, CA, USA) using an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA) as previously reported^{51 (link)}. Relative changes in transcript levels were normalized to the mRNA levels of GAPDH. The following primer sequences were used for real-time PCR analysis: OTX2, forward, 5′-ACAAGTGGCCAATTCACTCC-3′ and reverse, 5′-GAGGTGGACAAGGGATCTGA-3′; IRX1, forward, 5′-CGCGGATCTCAGCCTCTTC-3′ and reverse, 5′-CCCCAGGGTTGTCCTTCAGT-3′; BRACHYURY, forward, 5′-TGCTTCCCTGAGACCCAGTT-3′ and reverse, 5′-GATCACTTCTTTCCTTTGCATCAAG-3′; MIXL1, forward, 5′-CCGAGTCCAGGATCCAGGTA-3′ and reverse, 5′-CTCTGACGCCGAGACTTGG-3′; SOX17, forward, 5′-CGCTTTCATGGTGTGGGCTAAGGACG-3′ and reverse, 5′-TAGTTGGGGTGGTCCTGCATGTGCTG-3′; FOXA2, forward, 5′-TGGGAGCGGTGAAGATGGAAGGGCAC-3′ and reverse, 5′-TCATGCCAGCGCCCACGTACGACGAC-3′; Nestin, forward, 5′-GGCAGCGTTGGAACAGAGGT-3′ and reverse, 5′-CATCTTGAGGTGCGCCAGCT-3′; GAPDH, forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

cDNA samples were diluted 20 times for qRT-PCR analysis. PCR reactions were performed in an optical 384-well plate equipped with an ABI PRISM 7900 HT sequence detection system (Applied Biosystems) by using SYBR Green to monitor dsDNA synthesis, as previously described (Czechowski et al., 2005 (link)). The reaction mixture contained 4 μl of 2x Power SYBR Green PCR Master Mix reagent (Applied Biosystems), 2 μl of diluted cDNA, and 200 nM of each gene-specific primer in a final volume of 8 μl. The standard thermal profile of the manual was used for all PCR reactions. Amplicon dissociation curves, i.e., melting curves, were recorded. Data were analyzed using SDS 2.4 software (Applied Biosystems). PCR efficiency (E) was evaluated using a standard curve generated via PCR by using a fivefold dilution series.

DNA from the brain samples collected at 30 dpi was prepared. The DNA was extracted, purified, and quantified for parasite counts by real-time PCR using the B1 gene as previously described in detail [26 (link)]. PCR was performed using the ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA), and amplification was monitored using the SYBR green method (Applied Biosystems). The calculated cycle threshold (Ct) values were exported to Microsoft Excel for analysis. A standard curve was constructed with the T. gondii DNA extracted from 1 × 10⁵ parasites using 1 μl of a serial dilution ranging from 10,000 to 0.01 parasites. Parasite numbers were calculated by interpolation of a standard curve on which the Ct values were plotted against known concentrations of parasites. To confirm the specificity of the PCR, DNAs from the brain of an uninfected mouse and from purified T. gondii tachyzoites were used as the negative and positive controls, respectively.

Rs11057401 was genotyped using PCR-based Invader assay with the probe sets designed and synthesized by third wave. The genotyping results were read with an ABI PRISM7900HT sequence detection system (Applied Biosystems, Foster City, CA). Twenty percent of the samples were selected randomly to validate the reproducibility of the genotyping results.

Total RNA was extracted from the kidney, mesangial and podocyte cells using TRIzol reagent (Sigma‐Aldrich). cDNA (1.0 μg) was prepared from the total RNA using a commercially available kit (Toyobo). Next, quantitative real‐time PCR was performed using the SYBR Green PCR master mix (Applied Biosystems) on an ABI PRISM 7900HT sequence detection system (Applied Biosystems). The specific gene expressions were analysed using the following primers: mouse podocin: forward 5′‐AAG TGC GGG TGA TTG CTG CAG AAG‐3′, reverse 5′‐TGT GGA CAG CGA CTG AAG AGT GTG‐3′; mouse IL‐1β: forward 5′‐CAA CCA ACA AGT GAT ATT CTC CAT G‐3′, reverse 5′‐GAT CCA CAC TCT CCA GCT GCA‐3′; mouse sterol regulatory element‐binding protein‐1 (SREBP1): forward 5′‐GGA GCC ATG GAT TGC ACA TT‐3′, reverse 5′‐GCT TCC AGA GAG GAG GCC A‐3′; mouse fatty acid synthase (FAS): forward 5′‐AGA GAT CCC GAG ACG CTT CT‐3′, reverse 5′‐ GCC TGG TAG GCA TTC TGT AGT‐3′; mouse TGF‐β1: forward 5′‐CCT GTC CAA ACT AAG GC‐3′, reverse 5′‐GGT TTT CTC ATA GAT GGC G‐3′; mouse intercellular adhesion molecule (ICAM): forward 5′‐GGG ACC ACG GAG CCA ATT‐3′, reverse 5′‐CT CGG AGA CAT TAG AGA ACA‐3′; and mouse glyceraldehyde 3‐phosphate dehydrogenase: forward 5′‐TGA ACG GGA AGC TCA CTG‐3′, reverse 5′‐GCT TCAC CAC CTT CTT GAT G‐3′. ICAM and TGF‐β1 mRNA expressions were additionally confirmed to band using PCR.

Total RNA (500 ng) from each tissue sample was subjected to reverse transcription with oligodT, dNTPs, and M-MLV reverse transcriptase (Promega, USA). Primers for FoxC2 and GAPDH genes were designed for real time PCR analysis (Table S2). Quantitative RT-PCR was carried out as reported earlier [15] (link). The temperature conditions were as follows: 48°C, 30 min; 95°C, 10 min; followed by 40 cycles of 95°C,15 s; and 60°C, 1 min and analyzed using ABI Prism 7900HT sequence detection system (Applied Biosystems, CA). Values were normalized with GAPDH mRNA levels. A single peak was observed in the dissociation curve for both genes confirming the specificity of PCR products. Real time mRNA fold change was calculated by the formula, 2^−ΔΔCt [2^{(Ct value of target gene-Ct value of control gene)}].