6 protocols
normalization data transformation protocol
For each miRNA, the expression level was determined by the 2(-Delta Delta C(T)) method (Livak & Schmittgen, 2001). Data were normalized using the small nucleolar human RNA RNU48 as a control. 1st step of normalization: CT correction based on the global mean CT obtained between plates 2nd step of normalization: Target gene signal normalized to housekeeping gene RNU48 ID_REF = VALUE = normalized signals ('Data processing' steps 1/2)
array scanning protocol
hybridization protocol
labelling protocol
Reverse transcription reactions contained 390 ng of purified RNAs, 1x Megaplex RT primers (Human pool A v2.1, Applied Biosystems), 1x RT buffer, 2.5 mM of each dNTP, 3 mM MgCl2, 1U/µL of Multiscribe reverse transcriptase (Applied Biosystems) and 0.3 U/µL of RNase inhibitor (Applied Biosystems). The RT reaction had a final volume of 7.5 µL and was incubated for 5 min on ice, followed by 40 cycles (16°C 2 min, 42°C 1 min, 50°C 1 sec), 85°C 5 min and then held at 4°C. The RT products were subsequently amplified with sequence-specific primers contained in a TaqMan array MicroRNA card (card A, Applied Biosystems), which enables the simultaneous quantification of 377 different human miRNAs plus several endogenous controls. Six µL of RT products were diluted in 444 µL of nuclease-free water and mixed with 450 µL of 2x TaqMan Universal PCR Master Mix with no Amperase (Applied Biosystems) and dispensed into the 384 wells by centrifugation. The plates were incubated at 95°C for 10 min, followed by 40 cycles (95°C 15 sec, 60°C 1 min). The data were collected and processed using the Plate Utility and Automation Controller software (Applied Biosystems).
nucleic acid extraction protocol
Total RNAs, including small RNAs, were isolated using Qiazol reagent (Qiagen) in combination with the miRNAeasy kit (Qiagen) according to the manufacturer’s protocol. Purified RNAs were eluted in 30µL RNase-free water.
growth protocol
Confluent A549 cells were Mock-infected or infected with influenza viruses at an MOI of 1 or 10-3 for 1 h in a minimal volume of DMEM supplemented with 2 mM L-glutamine, 100 U/mL of penicillin, 100 µg/mL of streptomycin sulphate and 0.5 µg/mL of trypsin (infection medium) at 37°C. The cells were then overlaid with fresh infection medium and incubated at 37°C for 24 h.