7500 fast machine

Total RNA was extracted using a ReliaPrep™ RNA Cell Miniprep System (Promega) and quantified by a Nanodrop 2000 (Thermo, Wilmington, DE, USA). For RT-PCR, 0.5 μg of total RNA from each sample was used for cDNA synthesis using the GoScript™ Reverse Transcriptase system (Promega). Real-time PCR was performed on a real-time PCR system (7500 Fast Machine, Applied Biosystems, Thermo) using GoTaq^® qPCR Master Mix (Promega). The comparative Ct (∆∆Ct) method was used to calculate the gene expression levels after normalization, according to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The data show the relative values to the mRNA levels when cultured on control tissue culture plastic. The primers and their sequences are shown in Table 1.

RNA was isolated from gametocytes using an RNA purification kit (Stratagene). cDNA was synthesised using an RNA-to-cDNA kit (Applied Biosystems). Gene expression was quantified from 80 ng of total RNA using an SYBR green fast master mix kit (Applied Biosystems). All the primers were designed using the Primer3 software (Primer-blast; NCBI). Analysis was conducted using an Applied Biosystems 7500 fast machine with the following cycling conditions: 95°C for 20 s followed by 40 cycles of 95°C for 3 s; 60°C for 30 s. Three technical replicates and three biological replicates were performed for each assayed gene. The hsp70 (PBANKA_081890) and arginyl-t RNA synthetase (PBANKA_143420) genes were used as endogenous control reference genes. The primers used for qPCR can be found in Table S3.

An RNeasy purification kit (Qiagen) was used to isolate total RNA from purified parasites and an RNA‐to‐cDNA kit (Applied Biosystems) was then used to synthesis cDNA. SYBR green fast master mix (Applied Biosystems) was used for real‐time qRT‐PCR reactions and data were analysed using an Applied Biosystems 7500 fast machine with the following cycling conditions: 95°C for 20 s followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. Gene expression was determined using the Pfaffl method (Pfaffl, 2001) and used hsp70 and arginine‐tRNA synthetase as reference genes. Three biological replicates were used for each stage (each with two technical replicates). The primers used are described in Table S5. Statistical analyses (unpaired Student's t‐test) were performed using Excel and Grafit.

RNA was isolated using the QuickExtract RNA Extraction Kit (Epicentre, Inc.) and treated with DNase I. The SensiMix Probe One-Step Kit (BioLine, Inc.) along with single tube Custom TaqMan Gene Expression Assays (Applied Biosystems, Inc.) was utilized to carry out all real time polymerase chain reactions (RT-PCR). Reactions were run in duplicate on an Applied Biosystems 7500 Fast machine, according to the following cycling conditions: one cycle at 48 °C for 30 min, followed by 40 at 95 °C for 1 s, and 65 °C for 20 s. Means of the firefly: renilla RNA expression ratio was handled as in box in Fig. 1c.

RNA was extracted using TRI reagent (Applied Biosystems by Life Technologies), quantified using Nanodrop (Nanodrop 2000, Thermo Fisher Scientific, Waltham, MA, USA) and reverse transcribed using PrimeScript RT (Takara Bio Inc, Otsu, Japan). qPCR was performed with SYBR Green (Applied Biosystems by Life Technologies) on a 7500 Fast machine (Applied Biosystems). Samples were run in triplicates, and Actb or Gapdh were used as house-keeping genes. The delta-delta CT method was applied for relative quantification. Melting curves were obtained for each run. Primers were ordered from Microsynth (Switzerland), and sequences are shown in Table 1. All amplified products were run on agarose gels to verify proper amplification.

A nasopharyngeal swab was collected into universal transport medium (Diagnostic Hybrids, Athens, OH, USA) and stored at −80C. RNA was extracted using commercial kits (Qiagen), and specific RNA for rhinovirus, respiratory syncytial virus (RSV), human metapneumovirus (HMPV) and parainfluenza viruses (PIV) 1–4 was converted to cDNA and amplified by reverse transcriptase real‐time PCR (AgPath‐ID™ One‐Step RT‐PCR kit, Invitrogen) using primer/probe combinations as described 24 on a 7500 Fast machine (Applied Biosystems). A positive test was defined as Ct values ≤38.