Iq5 instrument

qRT-PCR was performed on Bio-Rad IQ5 instrument (Bio-Rad, Madrid, Spain) and MxPro Mx3005p qPCR thermal cycler (Stratagene, Madrid, Spain) using, respectively, SYBR Green (Bio-Rad, Madrid, Spain) and Dynamo SYBR Green (Finnzymes, Finland) master mix as described previously [44 (link), 45 (link)]. The primer pairs were located in different exons to rule out genomic DNA amplification. Each primer pair used yielded a single peak of dissociation on the melting curve and a single band with the expected size on PAGE gels. Identity of the PCR products was confirmed by sequencing. NT and non-RT RNA template reactions were used as negative controls. All PCR setups were performed at least in triplicate. Relative quantifications were calculated with the comparative ΔCt cycle method with normalization to the expression of housekeeping genes coding for ribosomal protein L19 (Rpl19), β-actin, glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and β-D-glucuronidase (Gusb). The efficiency of target and reference amplifications was tested to be approximately equal. miRNA qRT-PCR was performed using Exiqon LNA microRNA qRT-PCR primers and detection kit (Exiqon, Madrid, Spain) according to manufacturer's guidelines. All reactions were run in triplicate using 5S as normalizing control. Primer sequences and additional data are available upon request.

To investigate mRNA levels of CsACAT at different developmental stages of C. sinensis, total RNA was extracted from adult worms, metacercariae or eggs using TRIZOL reagent (Invitrogen, USA) according to the manufacturer’s instructions. cDNA synthesis was performed with First Strand cDNA Synthesis Kit (Fermentas, Canada) and oligo (dT)₁₈ primer by using 2 μg of total RNA. Relative mRNA levels were quantified on a BIORAD iQ5 instrument (BioRad, USA) by using 100 ng of cDNA with the SYBR Premix ExTaq Kit (TaKaRa, Japan) according to the recommendations. 5′-CGTCATCTGTGCGGGCGGTAT-3′ and 5′-TTGGCGTAGGCGTCCTGCTCT-3′ were employed to amplify specific fragments of CsACAT. β-actin from C. sinensis (Cs β-actin, GenBank accession No. EU109284) was used as an internal control (323 bp) [20 (link)]. The forward and reverse primers were 5′-ACCGTGAGAAGATGACGCAGA-3′ and 5′-GCCAAGTCCAAACGAAGAATT-3′. The real-time PCR procedure was as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 20 s. The melting curves were analyzed automatically by collection of the fluorescence signals. Bio-Rad iQ5 software was used to analyze the data according to 2^−ΔΔCt method [21 (link)].

Cells were cultured in 100 mm culture dishes (1 × 10⁶) for 24 h. After being pretreated with AI (100 μg/mL), CL (100 μg/mL), ACE (25 or 50 μg/mL), curcumin (5 μg/mL), or scopoletin (5 μg/mL) for 4 h, the cells were treated with 200 μM PA for 6 h. Total RNA from cells was extracted using the QIAzol reagent (Germantown, MD). Complementary DNA (cDNA) synthesis from the RNA was performed using a High-Capacity cDNA Reverse Transcription Kit (Ambion, Austin, TX, USA). Real-time PCR was performed on 4 genes including β-actin using an iQ5 instrument (Bio-Rad, USA). The primer sequences were as follows: CD36: 5′-GCC AAG CTA TTG CGA CAT GAT-3′ and 3′-GAA AAG AAT CTC AAT GTC CGA GAG T-5′, SREBP1c: 5′-GAG CGA GCG TTG AAC TGT AT-3′ and 3′-ATG CTG GAG CTG ACA GAG AA-5′, PPAR-γ: 5′-AGG TGG AGA TGC AGG TTC TA-3′ and 3′-TGG GAG ATT CTC CTG TTG AC-5′ and PPAR-α: 5′-TGG CAA AAG GCA AGG AGA AG-3′ and 3′-CCC TCT ACA TAG AAC TGC AAG GTT T-5′.

Primer sequences are shown in Additional file 8. Total RNA was extracted at time zero, 3 and 5 hours after induction of hyphal growth. On-column DNase digestion was performed according to the manufacturer’s instructions (QIAGEN). First strand cDNAs were synthesized from 1.6 μg of total RNA in 20 μl final volume, using the Reverse Transcription System A3500 (Promega). As a test for residual genomic DNA contamination, reactions were performed in the absence of reverse transcriptase. Real time quantitative PCR reactions were carried out in a Bio-Rad IQ5 instrument. Each PCR reaction contained 5 μl of diluted cDNA, 7.5 μl of SsoFast™EvaGreen^®Supermix with low ROX (Bio-Rad), 1.5 μl of oligos (final concentration each 0.5 μM) and 1 μl nuclease-free H₂O. Triplicates of all reactions were analysed. For quantification, the abundance of each transcript during induction conditions was determined relative to the standard TDH3 transcript, as indicated in other reports [79 (link), 80 (link)]. Final data on relative gene expression between the two conditions (sample from time 3 or 5 h with respect to time zero of each strain) and from two independent biological replicates were calculated according to the 2^-ΔΔCT method [81 (link)].

Histone variants mRNA quantities were assessed by qRT-PCR. 500,000 cells were harvested in each condition prior to total RNA isolation using a Nucleospin RNA XS kit (Macherey-Nagel). Reverse transcription was performed using random hexamer and oligo(dT)18 primers. Variant-specific histone primers were designed to quantify the total mRNA level and the PolyA subpopulation (listed in Table S1). Quantitative real-time PCR was performed on a Bio-Rad iQ5 instrument. The reactions were prepared using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen 11733-046). GAPDH was used as a control gene for normalization.

The specific primers for the eleven genes involved in carotenoid biosynthesis (PSY, PDS, ZDS, CRTISO, LCYb, CHYb, CYP, ZEP, NCED, CCD1, and CCD4) and two reference genes (26 S ribosome and Actin) (Supplementary Table 1) were designed on the basis of our RNA-Seq data (NCBI Sequence Read Archive: PRJNA506502). Gene transcript levels were detected using an iQ5 instrument (Bio-Rad Laboratories, Inc., United States) with the SYBR Premix Ex Taq II Kit [TaKaRa Biotechnology (Dalian) Co., Ltd., China]. The amplification procedure was as follows: 95°C for 1 min, followed by 40 cycles at 95°C for 20 s, 58°C for 20 s, and 72°C for 30 s. The Ct values of the reactions were recorded, and gene transcript quantification was performed based on the relative expression of the selected genes against that of Actin using the 2^–ΔΔCT method (Zhang et al., 2017 (link)). Three biological replicates were used for each analysis.