Mastercycler ep realplex real time pcr system

Total RNAs were isolated from JEG-3 cells using Trizol reagent (Invitrogen) and was reverse-transcribed into cDNA using the ReverTra Ace kit (Toyobo, Osaka, Japan) according to the manufacturer's instruction. Real-time quantitative PCR was carried out in a 20 μl reaction containing 0.6 μl of cDNA using the Eppendorf Mastercycler ep realplex real-time PCR system. After 15 min denaturation at 95°C, 40 cycles of amplification were carried out: 20 sec at 94°C, 10 sec at 58°C, and 15 sec at 72°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control in each sample and the amount of cDNA was normalized against that of GAPDH. Negative controls in which cDNA sample was replaced with PCR grade water for each primer pair were included in each run. Specificity was confirmed by melting curve analysis and agarose gel electrophoresis. The sequences of gene-specific primers were listed in Table 2.

Total RNA was extracted from white blood cells obtained from non-CHIKF febrile disease (n = 2), mild CHIKF (n = 3) and severe CHIKF (n = 2) patients using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH). Samples were treated with Dnase I (Promaga, Madison, WI) to remove genomic DNA before cDNA generation using oligo-dT (Bio Basic, Inc.) and Improm-II™ reverse transcriptase enzyme (Promega). The generated cDNA was use as template for quantitative real time PCR performed based on the SYBR system using thekAPA SYBR® FAST qPCRkit 2× Master Mix (Kapa Biosystems Inc., Woburn, MA) in a Mastercycler® ep realplex real-time PCR system (Eppendorf AG, Hamburg, Germany). Synthesis was carried out at an initial 95°C for 3 min followed by denaturation at 95°C for 10 seconds, annealing at 60°c for 30 seconds and extention at 72°C for 20 secs for 40 cycle using primers for caspase 1 (caspase-1fw: 5′-ACCAGGAAACGGAAACAGAGTGGT-3′ and (caspase-1rv: 5′-CTGCCCACAGACATTCATACA-3′) and actin (Actinfw :5′-ACCAACTGGGACGACATGGAGAAA-3′) and (Actinrv: 5′-TAGCACAGCCTGGATAGCAACGTA-3′). The relative expression levels of caspase 1 mRNA was normalized to actin using the comparative C_T method (2^-∆CT method). The fold change in expression between the non-CHIKF patients and CHIKF patients was calculated as 2^-∆CT (CHIKF patients)/ 2^-∆CT (non-CHIKF patients).

RNA extraction and cDNA synthesis from the liver and testis from POST-CTRL and BPA groups were conducted as previously described [60 (link)] with RNAzol solution (Sigma-Aldrich, Italy). For cDNA synthesis, the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) was used with 500-ng total RNA in a final volume of 100 µL.
Real-time quantitative PCR was carried out as previously described [60 (link)] with an Eppendorf Mastercycler Ep Realplex real-time PCR system (Eppendorf, Wesseling-Berzdorf, Germany), using two 96-well PCR-array layouts designed for the simultaneous profiling of a panel of 25 genes for gonad samples and 3 genes for liver samples, selected as: i) endocannabinoid system markers: cb1, cb2, pparα, pparβ, pparγ, faah, nape-pld, abdh4, cyt-pla2, cox2, dalgα, abdh6a, abdh12a, trpv1 ii) reproductive markers: era, erb, pr, ar, lhr, fshr, gnrhr, 17β-hsd, 3β-hsd, lepr, lep, vtg, zp1, zp3. PCR-array primers (Supplementary Table S1) were designed to obtain amplicons of 50–150 bp in length. A housekeeping gene (β-actin) and controls for general PCR performance were included on each array. The genes selected as well as the PCR results from POST-CTRL were the same from the previous published study from our group [44 (link)] to possibly compare the effects of the di-isononyl phthalate (DiNP) and BPA.

Two month-old lotus seedlings were used for submergence treatment and copper treatment. Seedlings were completely submerged or treated with 5 mm copper for 24 h (as described above) and then rhizomes were sampled. The sample without copper or submergence treatment was the control (Con). Meanwhile, different tissues of control seedlings were sampled. Tissues were homogenized with mortar and pestle in liquid nitrogen. Total RNA was isolated using CTAB-LiCl method. About 4 μg of total RNA was reverse-transcribed using an oligo(dT) primer and SuperScript Reverse Transcriptase (Invitrogen, USA). Real-time quantitative PCR was conducted using SYBR green (TaKaRa Biotechnology) on Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany) with a final volume of 20 μl per reaction. The specific primers were listed in Table S1. The relative transcript abundance was normalized using lotus Actin gene. Gene expression in various tissues were represented by NnTPS/Actin. Fold changes of genes under copper stress and submergence treatment were values relative to control samples after normalization to Actin transcript levels. RT-qPCR data presented were the means ± SE of at least four independent experiments. Differences among treatments were analyzed by on-way ANOVA, taking P < 0.05 as significant according to multiple comparisons or t-test.