Taqman probe

Bacterial strains were grown for five h (A₆₀₀ of 1.1 to 1.2) in low-glucose DMEM (Life Technologies). One ml aliquots of these bacterial cultures were mixed with two ml of RNA-Protect solution and the total RNA was isolated by using the RNeasy Kit according to the manufacturer's instructions (Qiagen). RNA was subjected to DNase treatment by using the Ambion TURBO DNA-free Kit to remove DNA contamination according to the manufacturer's instructions (Life Technologies). QRT-PCR was performed by adding 25 ng of DNase-treated RNA, 0.75 µM each of antisense and sense primers, 0.25 µM of a TaqMan probe (labeled at the 5′ end with FAM reporter and at 3′ end with TAMRA quencher) (Integrated DNA Technologies, Coralville, IA), to a QRT-PCR Master Mix (Agilent Technologies, Santa Clara, CA). Primers and labeled probes used for QRT-PCR are listed in Table 2. QRT-PCR was carried out in Mx3005P qPCR System (Agilent) by using the following parameters: 50°C for 30 min for cDNA synthesis; 95°C for 10 min; 35 cycles of 95°C for 30 sec, 55°C for 60 sec, and 72°C for 30 sec for cDNA amplification and detection of amplification products. The fold-change in the gene expression was plotted by adjusting the expression of a target gene to one for the parent (calibrator) strain and by normalizing to the expression of the house-keeping gene rpoA.

Reverse transcription was carried out using the SuperScript First-Strand Synthesis System for RT-PCR (Life Technologies) or ReverTra Ace (TOYOBO) following the manufacturer’s instructions. Total RNA (0.2–5 μg) was used for reverse transcription. Reverse-transcribed cDNAs were subjected to real-time PCR, which was performed with a LightCycler 480 Instrument (Roche Diagnostics) or CFX96 Touch Real-Time PCR System (Bio-Rad). For the detection of IER5, HSF1, HSPA1A and GAPDH, a TaqMan probe (IER5; Hs00275419, HSF1; Hs00232134_m1, HSPA1A; Hs00359163_s1, GAPDH; Hs02758991_g1)from Applied Biosystems were used. For the detection of HSPA6 and HSPA1B, a TaqMan probe from Integrated DNA Technologies was used. In some cases, custom-designed TaqMan Dual-Labeled Probes from Sigma-Aldrich were also used for the detection of HSPA1A and GAPDH.

Total RNA was prepared from gonad, heart, spleen, kidney, gills, intestine, hypothalamus and saccus vasculosus (BSH), and brain without BSH (brain) of six adult spring-spawning Atlantic herring using RNeasy Mini Kit (Qiagen). RNA was then reverse transcribed into cDNA with a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). TaqMan Gene Expression assay (ThermoFisher Scientific) containing 0.3 μM primers and 0.25 μM TaqMan probe (Integrated DNA Technologies) was performed to compare the relative expression levels of TSHR among different tissues. qPCR with SYBR Green chemistry was used for TSHB and DIO2 in a 10 μl reaction of SYBR Green PCR Master Mix (ThermoFisher Scientific) and 0.3 μM primers, with a program composed of an initial denaturation for 10 min at 95 °C followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Ct values were first normalized to the housekeeping gene ACTIN, then the average expression for each gene in the gonad was assumed to be 1 for the subsequent calculation of the relative expression in other tissues.

PCR screening for O. volvulus DNA was performed on all the infected mouse tissues to identify the presence of current or past O. volvulus larvae in that location. Mice were anesthetized and exsanguinated, and the internal organs were removed, and the skin was removed from the muscle. The muscle and skin were then divided into 100 different sections and individually frozen in 1.7 ml Eppendorf tubes. DNA was extracted from the tissue sections using the Promega genomic DNA kit A1125 following the manufacturer’s directions. Realtime PCR was performed using custom Taqman probes (Integrated DNA Technologies, Coralville, IA) against the Ov-150 [44 (link)–46 (link)] repeats and an ABI OneStep-Plus (ThermoFisher).

RNA was isolated from crude lysates or ribosomal fractions with QIAzol, following the manufacturer’s instructions. 2–4 ng of RNA were treated with 4 units of DNAse for 20 min at 37°C and inactivated for 15 min at 75°C. The RNA was converted to cDNA using SuperScript IV (ThermoFisher) with a mixture of Random primers and dT15V primers. 1 µl out of 10 µl of the resulting cDNA was used for ddPCR (Bio-rad) with Taqman probes (Integrated DNA Technologies; see key Resources table for the sequences) following the manufacturer’s instructions. The specificity of the primers for the HA-tagged versions of HRI was confirmed by ddPCRs in non-transfected neurons. The mRNA counts of each gene were normalized to the rRNA content of the corresponding fraction (ddPCR for rRNA was performed with cDNA diluted 1:10000). The normalized mRNA content of the fractions 7 to 11 (polysome) was related to the fractions 2–4 (monosome) with the following formula: (mRNA polysome)/(mRNA monosome + mRNA polysome) The disome was excluded from the calculations as its role in mRNA translation is not clear.