Nd 1000 spectrophotometer

Total cellular RNA was extracted from bladder cancer cells by using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. After quantified by a Nano Drop ND-1000 spectrophotometer, 500 ng RNA was reversely transcribed into cDNA according to the instructions provided by Takara reverse transcription kit (Takara, China). The resulting cDNA was amplified by SYBR Premix Ex Taq II (Takara, China) conducted on the Mx3000P instrument (Stratagene, USA). All the primers included in this study were provided by Invitrogen (Shanghai, China) and listed in Additional file 1: Table S2. The relative expression of target genes’ mRNA was calculated with the 2^-ΔΔCt method. GAPDH was used as internal control. All experiments were done in triplicate.

Viral nucleic acid was isolated from supernatant collected from virus infected cell culture using the TIANamp DNA Extraction Kit (Tiangen Biotech Co. Ltd., Beijing, China) (for viral DNA) or Trizol Reagent (Invitrogen, Beijing, China) (for viral RNA) according to the manufacturers’ instructions. C. psittaci nucleic acid was extracted from C. psittaci infected McCoy fibroblast cell lysate using the TIANamp DNA Extraction Kit. Nucleic acid was quantified using a ND-1000 spectrophotometer (NanoDrop, Wilmington, USA). One μg of FCV RNA was reverse transcribed using the Primescript II 1st strand cDNA Synthesis Kit (Takara Bio Inc., Dalian, China) according to the manufacturer’s instructions. The synthesized cDNA was purified using the cDNA Purification Kit (Takara Bio Inc.) and quantified using a ND-1000 spectrophotometer. All DNA and cDNA templates were stored at -20°C until assays were performed.

Quantitative RT-PCR assay was performed as described (81 (link)). Briefly, total RNAs from cultured cells were extracted with RNAiso Plus (Takara) according to the manufacturer’s instructions and the concentration was determined by a Nanodrop ND-1000 spectrophotometer. The RNA was transcribed into corresponding deoxyribonucleic acid (cDNA) using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (Takara). Each reaction was composed of the following mixture: 1 μl cDNA, 6.25 μl TB Green Premix Taq Ⅱ (2× ), 0.4 μl of each primer, and 4.45 μl ddH₂O was incubated in LightCycler480 real-time PCR system (Roche). The PCR protocol used was 95 °C for 30 s; 40 cycles of 95 °C for 5 s, and 60 °C for 20 s. All amplifications were done in technical duplicate and biological triplicate, and data were analyzed using LightCycler 96 SW 1.1 software. Primers are listed in the Table. S1C.