Express one step sybr greener kit

The gene expression levels of C. vicina pupae were analysed by amplification of molecular age markers, two for each of 15 developmental stages (labelled A1/A2–O1/O2) originally described in Zajac et al. [19 ] with the exception of markers E1 and E2, due to the small quantitative differences in expression (fold change values < 10). For markers D1 and D2, new primers were designed and established in comparison to Zajac et al. [19 ] for better performance (Supplementary Information, Table S2). The target sequences of the markers and the reference gene were annotated using blastx (Basic Local Alignment Search Tool). Almost all markers show homologies to the blow fly Lucilia cuprina, the closest relative with published transcriptome, but their functions are not yet known in detail. (Supplementary Information, Table S3). The validation of the molecular age markers was carried out by one-step RT-qPCR. The RT-qPCR was performed using the EXPRESS One-Step SYBR™ GreenER™ Kit, universal (Invitrogen™, Thermo Fisher Scientific) according to the manufacturer’s protocol in a reduced reaction volume of 10 µl, containing 40 ng total RNA, 100 nM ROX Reference Dye and 200 nM of each primer. For amplification, the standard cycling programme of the kit was performed on a 7500 Real-Time PCR System (Applied Biosystems™, Thermo Fisher Scientific).

Quantitative real-time PCR (qRT-PCR) was used to determine whether the ChIP experiment had worked prior to library construction and to validate both the ChIP-seq and RNA-seq data. To measure the enrichment of TF-binding targets in the immunoprecipitated DNA samples, 1 µl of IP or mock-IP DNA was used with Quantitect SYBR Green (QIAGEN) together with specific primers to the upstream region of Rv3616c, known to be bound by CRP^Mt. To validate the RNA-seq data, specific primers were used to quantify the messenger RNA targets showing up- or downregulation, and control targets showing no differential expression. RNA was extracted as described above, and 30 ng total RNA from wild-type and Δcrp cells was used with the Express One-Step SYBR GreenER kit (Invitrogen) according to the manufacturer's guidelines. All primer sequences and qRT-PCR results are in Supplementary Tables S1 and S2.

An aliquot of 2 × 10⁶/mL of N. crassa conidial suspension was directly inoculated into 100 mL liquid Vogel’s with 2% Avicel as carbon source in 250-mL Erlenmeyer flasks and cultured at 25°C in constant light with rotation (200 rpm) for 120 h. Biological triplicates were prepared for each strain. Total RNA was isolated from samples taken at 48, 96 and 120 h post-inoculation using zirconia/silica beads and a Mini-Beadbeater with 1 mL TRIzol reagent (from Invitrogen). RNA purification and DNase I treatment were performed using RNeasy Mini Kit (from QIAGEN). qRT-PCR was performed using EXPRESS One-Step SYBR^® GreenER™ Kit (from Invitrogen) on the CFX Connect™ Real-Time PCR Detection System (from Bio-Rad). Primer sequences used for amplification are described in Additional file 5: Table S2. The relative transcript level of major cellulase genes cbh-1 (NCU07340), cbh-2 (NCU09680), and gh5-1 (NCU00762) were normalized to expression from the actin gene (NCU04173) and calculated by $2^{-\mathrm{\Delta }{C}_{\text{t}}}$ as a relative expression level.

The CHIKV RNA copy number was quantified by RT-qPCR targeting the envelope E2 gene. Viral RNA copies were assessed using a standard curve of 10-fold serial dilution of a synthetic CHIKV RNA transcript [26 (link)]. One-step RT-qPCR was performed using EXPRESS One-Step SYBR GreenER Kit (Invitrogen, France) in a volume of 20 μL containing 10 ng (2 μL) of RNA template, 10 μL EXPRESS SYBR GreenER SuperMix Universal, 200 nM of sense Chik/E2/9018/+ and anti-sense Chik/E2/9235/− primers (Table 1) and 0.5 μL EXPRESS Superscript Mix. Amplification was performed on a LC480 LightCycler (Roche, France) and consisted of 15 min at 50°C followed by 95°C for 2 min, then 40 cycles of 95°C for 15 s and 63°C for 1 min. All PCR reactions were performed in triplicate and RNA from CHIKV-uninfected C6/36 cells was used as negative control.

Fluorescent probe 2,7-dichlorofluorescein diacetate (DCF-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT), triton X-100, FBS, and rhodamine 123 were purchased from Sigma (St Louis, MO, USA). DMEM-F12 was purchased from Gibco (Gibco, Grand Island, NY, USA). Caspase-3 Detection Kit was provided from Sigma. Express One-Step SYBR GreenER Kit was purchased from Invitrogen (Carlsbad, CA).

Mycelia were harvested by filtration and flash frozen in liquid nitrogen. RNA was extracted using the Trizol method (Invitrogen) and further purified using RNeasy kits (QIAGEN). Four ng of RNA was used as template in each quantitative RT-PCR (qRT-PCR) reaction. qRT-PCR was carried out using EXPRESS One-Step SYBR GreenER kit (Invitrogen) and Applied Biosystems Step One Plus Real Time PCR system. qRT-PCR were done in biological duplicates or triplicates with actin as the endogenous control. Relative expression levels were normalized to actin, and fold changes in RNA level were the ratios of the relative expression level on inducing conditions to no carbon conditions.