Rotor gene q

Six-point standard curves for each gene were prepared from a serial dilution of pooled cDNA, as described in [36 (link)]. Gene expression was assayed in Rotor-Gene Q (Qiagen), 72-well rotor, using FastStart SYBR Green Master kit (Roche), 3 μl 35×diluted cDNA samples and the recommended thermal profile (40 cycles). This was followed by a melt curve analysis and gel electrophoresis of the qPCR products. For each target gene, all of the cDNA samples, standards and no template controls (reactions without cDNA) were assayed in triplicate in a single run. The standard curve calculation and data analysis was performed with Rotor-Gene Q software (Qiagen). The following primers were designed with Primer-BLAST: for Ref A (125 bp amplicon), forward: 5’-CCTTTGCTCAGGGCTTGCCAGA-3’ and reverse: 5’- AACGCGGATCCTATGATGGCAC-3’; for Ref B (193 bp amplicon), forward: 5’- AGAGCGGTTTGTGTTCTCCTTGG-3’ and reverse: 5’- GGGGGTGGGCAACAACGCTT-3’; for Ref C (110 bp amplicon), forward: 5’- ATACCCTGCCTTCACTGCGTGT-3’ and reverse: 5’- TGAGACCAGGCTTCCTTGGGATT-3’; for Ref D (156 bp amplicon), forward: 5’- TGTCTGCGGGTAGAAGAAGTGGA-3’ and reverse: 5’- GCTGAGTTACTCCCCTCACTCACA-3’; and for Ref E (108 bp amplicon), forward: 5’- CCACAAGTAGGCACTTCAGGTAG-3’ and reverse: 5’- CCTGTGAGATGTGGAATCCGCTGC-3’.

In order to determine if a pool was positive for IAV, a previously published real-time RT PCR assay targeting the matrix gene of IAV [51 (link)] was adopted. Briefly, the published primers and the OneStep RT-PCR kit (QIAGEN, Copenhagen, Denmark) was used for the PCR mix, which was subsequently run on the Rotor-Gene Q (QIAGEN, Copenhagen, Denmark) using the following program: 50 °C, 30 min; 95 °C, 15 min; cycling 45× (95 °C for 10 s, 60 °C for 20 s, 64 °C for 1 sec, 68 °C for 1 sec, 72 °C for 30 s). All samples were tested in duplicates and the pool was only considered positive if both samples gave a positive result and had a Ct value <36. All positive samples with a Ct value <31 were tested to determine the IAV subtype using the previously published multiplex real-time RT PCR assay [52 (link)] with the modifications described in a previous study [45 (link)] and run on the Rotor-Gene Q (QIAGEN, Copenhagen, Denmark). The individual samples of two most positive pools of each sampling were also tested. Positive individual samples were selected for viral isolation and sequencing.

RNA was extracted from swabs, blood, and CSF using the QIAmp Viral RNA kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Tissues (30 mg) were homogenized in RLT buffer and RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions. RNA was eluted in 50 µl total volume. Bornaviral RNA was detected by RT–qPCR using a VSBV-1 specific (Squirrel mix 10.1) (25 ) or a BoDV-1 specific assay (BoDV-1 mix 1) (17 (link)) and the qScript XLT one-step RT-qPCR ToughMix Kit (Quanta BioSciences, Gaithersburg, MD, USA). In each run, standard dilutions of quantified RNA standards were run in parallel, to calculate copy numbers in the samples. All RT-qPCRs were performed on a Rotorgene Q (Rotorgene Q software version 2.3.1; Qiagen). Dideoxy chain-termination sequencing for VSBV-1 and sequence analysis was done as described elsewhere (3 (link), 25 ). For BoDV-1, primers are given in Table S1 (Supplementary Material).

Tissue samples were collected, immediately frozen in liquid nitrogen, and stored at −80 °C until use. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany) and served as template for cDNA synthesis. From each sample, two independent cDNA syntheses from 250 ng total RNA were performed using SuperScript III (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was carried out on a Rotor Gene Q (Qiagen Hilden, Germany) by using TaqMan technology with various fluorescent dyes to allow duplex measurements of receptor and reference gene expression. Fluorescent dyes used as 5′-modifications were 6-FAM-phosphoramidite (6FAM), Cy5, Cy5.5 and Yakima Yellow (YAK). BlackBerry quencher (BBQ) was attached to the 3′-end of TaqMan probes. The sequences of the primers and TaqMan probes are presented in Table 1. The PCR was performed with an initial step at 60 °C for 1 min and a denaturation step at 95 °C for 5 min, followed by 45 cycles at 95 °C for 20 s and 60 °C for 60 s. Tissue samples of individual bees were examined in triplicate. Mean copy numbers were calculated using Rotor Gene Q software (Qiagen). Receptor transcript levels were normalized to elongation factor 1α (Amef-1α) transcript levels (=100%) using the standard curve method. The standards covered copy numbers from 10⁴ to 10⁷.

All qPCR runs followed a maximum sample layout for each gene (all tissue samples included in one run) and comply with the MIQE guidelines [32 (link)]. Each run also included no template controls (NTC) for each primer pair and three inter run calibrators (IRC). EF1α, RNAP and UBI were randomly chosen as IRCs and were run on mixed tissue cDNA. Samples were run in triplicate, NTC and IRCs were run in duplicate. qPCR reactions contained 5 μL 2x Rotor-Gene SYBR Green PCR Master Mix (Qiagen, #204074), 0.4 μL cDNA, 0.1 μL of each primer (1μM final concentration) and 4.4 μL ddH₂O to a final volume of 10 μL. Amplification and detection were performed on a Rotor-Gene Q (Qiagen) running Rotor-Gene Q software (version 2.0.2) with the following temperature profile: 5 min initial activation and denaturation at 95°C; 40 cycles of 5 sec denaturation at 95°C, 10 sec annealing and extension at 60°C (data collection at this step); a final melt curve analysis from 60°C to 95°C at a rate of 5 sec/1°C. PCR amplicons were also assessed on agarose gels to qualitatively visualise amplification efficiency (S1 Fig).

Total RNA from ARPE-19 cells was prepared from 3 subconfluent wells of a 6 well plate. Cells were harvested by accutase (Sigma) and RNA was subsequently isolated using RNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was reverse transcribed using Quantitect Reverse Transcription Kit (Quiagen) according to the manufacturer’s protocol. The mRNA levels of ANO4 (hsAno4_F: 5′-TTGTAAGGCGGTACTTTGGAGA-3′ and hsAno4_R: 5′-ATCCAGAGTGGTGACGCCATA-3′) and the housekeeping gene GAPDH (hsGAPDH_F: 5′-TCAACGACCACTTTGTCAAGCTCA-3′; hsGAPDH_R: 5′-GCTGGTGGTCCAGGGGTCTTACT-3′)were compared in scRNA and siRNA transfected ARPE-19 cells by Real Time RT-PCR using SYBR Green PCR Master Mix (Quiagen) on an Rotor-Gene Q (Qiagen). Experiments were repeated four times. Data were analyzed with the Rotor-Gene Q software 2.1.0 (Qiagen). According to Willems et al.^{27 (link)} the ddCt values went through a series of sequential corrections, including log transformation, mean centering, and autoscaling, to draw statistically reliable conclusions.