Prism 7500 fast real time pcr system

To verify the expression profile of the differentially expressed genes selected using bioinformatics method, we have performed quantitative real-time PCR (qRT-PCR). Total RNAs were reverse-transcribed using reverse transcriptase (Invitrogen, Carlsbad, CA, USA). All primers were designed using Primer Premier 6. The SYBR Green I Master Mix (TaKaRa, Dalian, China) was used for qRT-PCR using an Applied Biosystems Prism 7500 Fast Real-Time PCR system. One denaturation cycle was performed at 95 °C for 5 min, and 40 amplification cycles were performed at 95 °C for 15 s and 60 °C for 30 s. Results were expressed relative to the expression levels of beta-actin in each sample using the Relative Expression Software Tool (REST) version 2009. Expression differences between groups were assessed for statistical significance using R software. All samples were run in triplicate. The averages of three relative quantities of biological replications were used in a two-tailed Student’s t test with a 95 % confidence level (P < 0.05) to determine the significance with respect to gene expression.

Total RNA was isolated by using Qiazol (Life Technologies, Carlsbad, CA) and purified with the RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse-transcription and quantitative PCR assays were performed using High Capacity cDNA Archive kit and KAPA SyBr Fast qPCR kit (KAPA Biosystems, Wilmington, MA). For quantification of mRNA levels, 18S level was used as an internal control. All reactions were analysed in an Applied Biosystems PRISM 7500 Fast Real-Time PCR system in 96-well plate format. Real-time primer sequences shown in Supplementary Table 2.

Total RNA was isolated from C₂C₁₂ cells using TRIzol reagent according to the manufacturer’s protocol. cDNA was synthesised using a High-Capacity cDNA Reverse Transcription Kit. Gene expression was measured through quantitative PCR (ABI Prism 7500 Fast Real-Time PCR System) and analysed using 7500 Fast Sequence Detection Software v.1.3.1 (Applied Biosystems Inc., Foster City, CA, USA) using specific mouse TaqMan genes and Double delta Ct method. TaqMan genes: FABP4 (Mm00445878_m1), PPAR-γ (Mm00440940_m1), and the endogenous control GAPDH (Mm99999915_g1).

L3/young L4 nematodes of 500–1000 worms were dispersed in 6 cm Petri dishes after exposure to different TiO₂ NP concentrations (untreated control, 0.01, 0.1, 1, and 10 mg/L). The nematodes were gathered, and total RNA was extracted from each exposed sample using the TRIzol Reagent (Gibco, Life Technologies, Carlsbad, CA, USA).
To test for cDNA synthesis, 1000 ng of RNA was reversed to cDNA using a High-Capacity cDNA Reverse Transcription kit (Gibco, Life Technologies, Carlsbad, CA, USA). The real-time PCR was analyzed using the following processes. cDNA of 50 ng was amplified using the SYBR green PCR master mix (Gibco, Life Technologies, Carlsbad, CA, USA) for a 10 min 95 °C denaturation stage, 40 15 sec repetitions at 95 °C, and finally 1 min at 60 °C using an Applied Biosystems PRISM 7500 fast real-time PCR system. To measure the gene expression of the nematodes, quantitative methods were used to measure the expression of superoxide dismutase-1 (sod-1), sod-3, cyp-35A2, catalase-1 (ctl-1), ctl-2, melatonin-1 (mtl-1), mtl-2, and actin-1 mRNA. The sequences for the primers are shown in Table 3.

Total RNA samples were reverse transcribed into cDNA using random primers and an RNA PCR Kit (AMV) Ver.3.0 (Takara Bio Inc.). CXCL1, CXCL8, and chemokine (C-C motif) ligand (CCL) 2 levels were measured using the Power SYBR Green PCR Master Mix (Applied Biosystems). The following primers were used for analyses: 5′-TGC AGG GAA TTC ACC CCA AG-3′ and 5′-CAG GGC CTC CTT CAG GAA CA-3′ for CXCL1; 5′-ACT CCA AAC CTT TCC ACC CCA-3′ and 5′-TTT CCT TGG GGT CCA GAC AGA-3′ for CXCL8; 5′-CTT CTG TGC CTG CTG CTC AT-3′ and 5′-CGG AGT TTG GGT TTG CTT GTC-3′ for CCL2; 5′-GAA GGT GAA GGT CGG AGT CA-3′ and 5′-GAG GTC AAT GAA GGG GTC AT-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The Prism 7500 Fast Real-time PCR system (Applied Biosystems) was used for analyses, and the mRNA levels of the genes tested were represented as relative values to the expression level of GAPDH.

Total RNA was isolated by using TRIzol (Invitrogen) and purified with the RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse-transcription and quantitative PCR assays were performed using High-Capacity cDNA Reverse Transcription Kit and KAPA SYBR Fast qPCR kit (KAPA Biosystems, Wilmington, MA). For quantification of mRNA levels, ACTB or GAPDH level was used as an internal control. All reactions were analyzed in an Applied Biosystems PRISM 7500 Fast Real-Time PCR system in 96-well plate format. Primer sequences for specific genes are shown in Supplementary Table 1.