Cfx manage software

Viral RNA was extracted from patient NP samples using Qiagen QIAamp Viral RNA kit. Reverse-transcription and amplification are both performed in the same reaction using Quanta qScript XLT One-Step RT-qPCR ToughMix, with no ROX reference dye (95134-100). Taqman primer/probes (FAM) were purchased from Life Technologies these were designed to target the RSV nucleocapsid: RSV type-A (all 5′-3′: Forward - GCT CTT AGC AAA GTC AAG TTG AAT GA, Reverse - GCC ACA TAA CTT ATT GAT GTG TTT CTG, Probe - ACA CTC AAC AAA GAT CAA CTT CTG TCA TCC AGC) and RSV type-B (all 5′-3′: Forward - GAT GGC TCT TAG CAA AGT CAA GTT AA, Reverse - TGT CAA TAT TAT CTC CTG TAC TAC GTT GAA, Probe - TGA TAC ATT AAA TAA GGA TCA GCT GCT GTC ATC CA)^{11 (link)}. The RNA was run on CFX96^TM Real-Time system, C1000 Touch Thermal Cyler by Bio-Rad. Parameters were set for an initial run at 50 °C for 12:00 minutes (1), 95 °C for 1:00 minute (2), 95 °C for 7 seconds (3), 60 °C for 45 seconds (4), a plate read (5), then a repeat from 3, 45 times. Quantification of FFU/mL from C_T value for each virus is derived from a standard curve of a prototypical laboratory strain RSV type A2 virus of known infectivity run in parallel with all samples during each PCR run. C_T thresholds were computed for each run by the Bio-Rad CFX Manage software (Version 3.1) based on a control curve that was run in parallel with each plate run.

A reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assay was conducted according to our previously reported protocol [18 (link)]. Briefly, MCF-7 cells were seeded in six-well plates at a density of 5 × 10⁴ cells/well. After 24 h of incubation, T1 or T6 (0.2 μM) was added, and the cells were further incubated for 48 h. Total RNA was isolated using 1 mL of TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the PrimeScript RT reagent kit with gDNA eraser (code DRR047A; TaKaRa). The RT-qPCR was performed using SYBR Premix Ex Taq II reagent (code DRR081A; TaKaRa) with the standard cycling program (40 cycles of 95 °C for 30 s and 60 °C for 5 s), and melting curves were generated by a temperature increase from 65 °C to 95 °C at a rate of 0.5 °C/5 s using a CFX96 real-time PCR detection system (Bio-Rad Laboratories Ltd., Hercules, CA, USA). The forward and reverse primer sequences for RRM2 are shown in Table 1. The calculations were performed using the CFX Manage software (version 1.0; Bio-Rad Laboratories Ltd.). The results are expressed as fold changes in expression [2^(−ΔΔCt)] on a linear scale.

Total RNAs as described for RNA-Seq library construction were used for qRT-PCR. Reverse transcription was performed using 1 μg total RNA for each sample and 200 U M-MLV Transcriptase (TaKaRa) in a 10 μl volume. The reaction was conducted at 70 °C for 10 min, 42 °C for 60 min and 70 °C for 15 min. The resulting cDNA was diluted to 800 μl with sterile water. The qRT-PCR was conducted in triplicate reactions using the BIO-RAD CFX system (BIO-RAD). Gene-specific primers were designed by Primer3 (http://bioinfo.ut.ee/primer3/), and the primers for randomly selected 28 genes used in this study are listed in Additional file 16. The primers for validation of all the genes that described in the result part are listed in Additional file 17.
The 40S ribosomal protein S8 (c42687_g2) was used as the internal control for qRT-PCR because the expression level of this gene is relatively stable in samples of different tissues and different time points. The PCR was conducted in a 20 μl volume containing 4 μl diluted cDNA, 250 nM forward primer, 250 nM reverse primer, and 1 × SYBR Premix Ex Taq II (TaKaRa) using the following conditions: 95 °C for 3 min, 40 cycles of 95 °C for 15 s, 59 °C for 15 s and 72 °C for 15 s. Melting curve analyses were performed to verify the specificity using the Bio-Rad CFX Manage software. The relative expression levels were calculated using the 2^-ΔΔCT method [65 ].