Redtaq readymix pcr reaction mix

Human XBP1s and XBP1u mRNA levels were monitored by semi-quantitative real-time PCR using cDNA synthesized from dsDNase-treated RNA as described above, RedTaq® ReadyMix™ PCR Reaction Mix (Sigma) and the following primers: 5′- TAA TAC GAC TCA CTA TAG GGG AAT GAA GTG AGG CCA GT-3′ and 5′-AAT CCA TGG GGA GAT GTT CTG GAG-3′. For ACTB mRNA levels, the following primers were used: 5′-TTG CCG ACA GGA TGC AGA AGG A-3′ (fwd) and 5′-AGG TGG ACA GCG AGG CCA GGA T-3′ (rev). PCR products were resolved by agarose gel electrophoresis. Densitometric analysis was performed using Fiji software (version 1.47i) and corrected by subtraction of the appropriate background values.

Spatial-temporal expression patterns of the different CqCPAP3 genes in Cherax quadricarinatus were examined using RT-PCR. Total RNA was isolated from the molar-forming epithelium, carapace cuticle-forming epithelium, gastrolith-forming epithelium, hepatopancreas and abdominal muscle from males in four different molt-stages: inter-molt, early pre-molt, late pre-molt and post-molt; EZ-RNA Total RNA Isolation Kit (Biological Industries, Beit Haemek, Israel) was used according to the manufacturer’s protocol. First-strand cDNA was synthesized by reverse transcription using the qScript cDNA Kit (Quanta BioSciences, Gaithersburg, MD) with 1 µg of total RNA. Specific primers were used for PCR amplification (Table S1); PCR was performed with REDTaq ReadyMix PCR Reaction Mix (Sigma); using the following specific conditions: 94 °C for 1 min, followed by 35 cycles 94 °C for 1 min, 60 °C for 2 min, 72 °C for 3 min, followed by 10 min at 72 °C. Cq18S amplification served as a positive control.

For reverse transcription, 500 ng of isolated RNA was subjected to cDNA synthesis using SuperScript® III First-Strand Synthesis system (Thermo Scientific, USA) following manufactures instructions. 1ul cDNA was used as template for gene amplification. For PCR amplification of viral genes, REDTaq^® ReadyMix^™ PCR Reaction Mix (Sigma Aldrich, USA) was used. RSV N, M and NS1 genes were amplified using specific primers (sequence available upon request) that amplify both viral and corresponding antigenome sequences, under following thermal conditions: denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min for 35 cycles and a final extension step at 72 °C for 7 min. The PCR product were subjected to agarose gel (1.5%) electrophoresis and analyzed under UV trans-illuminator. To determine whether exosomes would carry RSV specific genome and/or antigenome sequences, we used primers targeting intergenic regions of RSV genome as previously described^{86 (link)}.
To validate the upregulation of miRNA expression in RSV exosomes, miRNA expression was examined using miScript II cDNA synthesis reagents, miRNA primers and miScript SYBR Green PCR system (all from Qiagen Inc.), following manufactures instructions.

Total RNA was extracted from B16-F10 and HBME cells using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. Then, 500 ng of total RNA were used to synthesize cDNA using SuperScript™ First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Next, cDNA was used for reverse transcription or qPCR using REDTaq ^® ReadyMix™ PCR Reaction Mix (Sigma-Aldrich, Saint Louis, MO, USA) or 5x HOT FIREPol^® EvaGreen^® qPCR Supermix (Solis BioDyne, Tartu, Estonia), respectively. The following primers were used: mouse β-actin (Internal control), forward: 5’-AGCTTCTTTGCAGCTCCTTC-3’, reverse: 5’-CCACCATCACACCCTGGT-3’; 14 kDa hGH, forward: 5’-CAGTGCCTTCCCAACCATTC-3’, reverse: 5’-GGAGCAGCTCTAGGTTGGAT-3’. The relative expression of the 14 kDa hGH gene was calculated according to the $2^{-\Delta \Delta C\left(t\right)}$ method. The reverse transcription and qPCR were performed in three separate wells for each condition.