Sensifast cdna synthesis kit

Human MDMs and J774 macrophages were infected with L. major stationary-phase promastigotes at a parasite-to-cell ratio of 10-to-1, for 4 h, after which non-internalized parasites were removed through extensive washing in PBS. Cells were then treated with a TDO-specific inhibitor (680C91, Sigma Aldrich) at a concentration of 20 μM, previously determined as non-toxic for both parasite and cells, or DMSO (vehicle). At 48 h after incubation at, cells were lysed in 800 μL of TRIzol reagent (Thermo Fisher Scientific. RNA was purified and reverse transcription and quantitative PCR using SensiFAST cDNA synthesis kit (Bioline) followed by a qPCR (SensiFAST SYBR Hi-ROX kit, Bioline). For gene amplification, mixtures were composed of SensiFAST buffer (2X, Bioline) and 200 nM of forward and reverse primer. Assays were performed in 10 μl reactions volume with 15 ng of cDNA sample. Thermocycling settings consisted of one hold of 2 min at 95°C followed by a two-step temperature (95°C for 15 s and 60°C for 30 s) over 40 cycles in a CFX384 touch real time PCR detection system (Bio-rad). To calculate the relative intracellular parasite growth between DMSO-treated and TDO-inhibitor treated cells, we quantified the parasite-specific transcripts KMP11 and RRNA45, using GAPDH and RPS18 as host reference genes.

qPCR assays were performed to confirm the RNA-seq results. One microgram of total RNA from each sample (the same samples as for RNA-seq) was used to synthesize cDNA using a SensiFAST cDNA synthesis kit according to the manufacturer’s instruction (Bioline, Inc. Cincinnati, OH, USA). The SensiFAST SYBR No-ROX kit was used for qPCR using a BIO-RAD CFX384 real-time PCR detection system (Bio-Rad laboratories), in accordance with the manufacturer’s protocols. The data were collected from three biological and three technical replications. Initially, we tested 5 reference genes including 18SrRNA, Elongation factor [Ef1α], GAPDH, Initiation factor [IF4a] and ACTIN. GAPDH was selected and used as a reference gene to normalize the relative quantities of the target genes because of its consistency across different time points and genotypes. The comparative CT method was used for quantification of gene expression, and fold change was calculated using 2^−∆∆CT [94 (link)]. The list of the selected genes and primers used for validation are listed in Table S11.

Intestinal tissues were collected and immediately stored in RNALater (Life Technologies). After removal of the RNALater, the tissues were homogenized using Trizol (Life Technologies) and 1.0-mm silicon carbide beads (Biospec Products) on the Precellys 24 at 4000 rpm for 30 s, three times. After centrifugation, the supernatant was collected and RNA extraction performed using a Direct-zol RNA purification kit (Zymo Research). cDNA was synthesized from tissue RNA (Sensifast cDNA synthesis kit, Bioline) and real-time PCR used the SensiFast SYBR Lo-ROX Kit (Bioline) on a ViiA7 real-time PCR system (Life Technologies). Data were analyzed in duplicates using the delta-delta Ct method, with HPRT as the housekeeping gene. Primers used are shown in Table 1.Table 1

Primers for qRT-PCR

IL-10	′5-CAGGGATCTTAGCTAACGGAAA-3′
	′5-GCTCAGTGAATAAATAGAATGGGAAC-3′
IL-22	′5-ATGAGTTTTTCCCTTATGGGGAC-3′
	′5-GCTGGAAGTTGGACACCTCAA-3′
HPRT	′5-CCTAAGATGAGCGCAAGTTGAA-3′
	′5-CCACAGGACTAGAACACCTGCTAA-3′
Lysoyzme P	′5-ATGGCTACCGTGGTGTCA-3′
	′5-CGGTCTCCACGGTTGTAGTT-3′
Cryptdin	′5-AAGAGACTAAAACTGAGGAGCAGC-3′
	′5-GGTGATCATGAGACCCCAGCATCAGT-3′

RNA was extracted from synchronized L3 and day-1 adult animals. Protocols for RNA extraction, cDNA synthesis and qPCR were described earlier⁷ . Briefly, total RNA was extracted using Trizol (Thermo Fisher, USA). The RNA was used to prepare cDNA and, subsequently, perform qPCR using the SensiFast cDNA synthesis kit (Bioline, USA), and SYBR green mix (Bio-Rad, Canada), respectively. Primers are listed in Table S4.

Quantitative real time PCR was conducted according to Liu et al. [109 (link)] to measure the gene expression of key transporters related to salt tolerance in rice. Total RNA from rice leaves was extracted in the 4th week after stress application using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed using a SensiFAST cDNA synthesis kit (Bioline, London, UK) according to the manufacturer’s instructions. The expression of genes (HAK1, NHX1, HKT1;4, SOS1, and VHA-c) known to be involved with salt tolerance was assessed by real-time quantitative RT-PCR using a Quantinova Sybr Green Kit (Qiagen, Valencia, CA, USA) in a Rotor-Gene 3000 quantitative PCR thermocycler (Corbett Research, Mortlake, NSW, Australia). G6PDH and Elfa were used as reference genes.

The genomic DNA fragment spanning from exon 5 to exon 6 of WSB1 gene was cloned into pcDNA3.1(+) plasmid and deletion mutants were generated by mutagenesis cloning using PrimeSTAR Max DNA Polymerase (Takara). Minigene plasmids were transfected into cells using lipofectamine 2000. After 48 h total RNA was purified using RNeasy Mini Kit (Qiagen) with RNAse-Free DNase Set (Qiagen) to digest contaminating DNA. Reverse transcription was performed using SensiFAST™ cDNA synthesis kit (Bioline) and PCR was performed using pcDNA3.1(+) forward and reverse primers by exTEN 2× PCR Master Mix (Axil Scientific). Primer sequences are listed in Supplementary data 7.