Rotor gene rg 3000 system

A. franciscana cysts were harvested within 1 day after release from females injected with dsRNA for either HSF1 or GFP. RNA was extracted from 80 cysts by homogenization with a micropestle (ThermoFisher Scientific) in a 1.5 ml microtube with 100 μl TRIzol^® (ThermoFisher Scientific). cDNA was generated with the SuperScript^® III First-Strand Synthesis Kit for RT-PCR (ThermoFisher Scientific) using 0.1 μg of RNA as template and oligo dT₂₀ primers. All RNA preparations were incubated without reverse transcriptase to ensure the absence of genomic DNA. qPCR was conducted with a QuantiTect^® SYBR Green PCR Kit (Qiagen, Mississauga, ON, Canada) in a Rotor-Gene RG-3000 system (Corbett Research, Sydney, NSW, Australia) using 1 μl cDNA and primers specific for HSF1, p26, ArHsp21, ArHsp22, artemin and tyrosinated α-tubulin at 10 μmol/L (Table 3). qPCR was at 50°C for 3 min and then at 95°C for 8 min followed by 30 cycles of 95°C for 20 s, annealing for 20 sec at the temperatures indicated in Table 3 for each mRNA, and 72°C for 40 s. mRNAs for proteins of interest were quantified in duplicate from three independently prepared RNA samples. Copy numbers of hsf1, p26, ArHsp21, ArHsp22 and artemin mRNAs were determined from a standard curve of Ct values and normalized against α-tubulin mRNA (King et al., 2013). Primer fidelity was assessed by melting curve analysis.

Total RNA from cultured cells was prepared using TRIzol reagent (Invitrogen),
followed by further purification through RNeasy mini-columns (Qiagen, USA) with
on-column DNase I treatment. cDNA samples were prepared by reverse transcription
using Accupower RT-pre mix (Bioneer,). Real-time PCR reactions were then
performed using the Rotor-Gene RG 3000 system (Corbett Research, Australia) with
diluted cDNA, SYBR qPCR master mixture (Kapa Biosystems, USA) as the reporter
dye, and 10 pmol of gene-specific primers. Thermal cycling conditions
comprised 95 °C for 3 min to allow for enzyme
activation, followed by 40 cycles at 95 °C for
10 s, 53 °C for 15 s and
72 °C for 30 s. The level of each mRNA was
normalized to that of GAPDH, and the values were plotted as mean±s.d.
of three independent experiments. Primer sequences used were as follows: for
α3, forward
(5-′AGAAGTGGAGCAGTTGATCA-3′) and reverse
(5-′TCTCTGATTCTATTTATCCTTTTCT-3′ for
endogenous α3, which targets 3′ UTR, or
5-′TCTCTGATTCTACTTGTCGTCATCG-3′ for
exogenous α3, which targets the flag tag); for Tau, forward
(5′-AAGGTGACCTCCAAGTGTGG-3′) and
reverse (5′-GGGACGTGGGTGATATTGTC-3′); for
α-Syn, forward
(5′-AAGAGGGTGTTCTCTATGTAGGC-3′) and
reverse (5′-GCTCCTCCAACATTTGTCACTT-3′);
for EGFP forward
(5′-ACGTAAACGGCCACAAGTTC-3′) and reverse
(5′-AAGTCGTGCTGCTTCATGTG-3′); for
GAPDH, forward
(5′-GAGTCAACGGATTTGGTCGT-3′) and reverse
(5′-GACAAGCTTCCCGTTCTCAG-3′).