RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- Include DNAse treatment for 15-20min - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C. - For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Protocol tips
- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
- Include DNAse treatment for 15-20min - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C. - For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Publication protocol

The siRNA duplexes for A549 cells were selected from Silencer select siRNA library (Ambion, Austin, TX). The siRNA duplexes for MDCK cells were synthesized (Sigma-Aldrich, St. Louis, MO). The sequences of siRNA duplexes for MDCK cells were listed in Table 1. A549 cells were seeded on type I collagen coated 96-well plate. Three target-specific siRNAs (Silencer select siRNA library, Ambion, Austin, TX) or non-targeting negative control siRNA (Silencer Negative Control #1 siRNA, Ambion, Austin, TX) at a final concentration of 10 nM were transfected using transfection reagent (Lipofectamine RNAiMAX; Invitrogen, Carlsbad, CA) and incubated at 37°C for 48 h. For MDCK cells, the cells were seeded on 96-well plate and three target-specific siRNAs (Sigma-Aldrich, Inc., St. Louis, MO) or non-targeting negative control siRNA (Silencer Negative Control #1 siRNA, Ambion, Austin, TX) at a final concentration of 50 nM were transfected by siRNA transfection reagent (Lullaby; OZ Biosciences, France). Cells were washed with PBS three times and infected with influenza A virus at a MOI of 0.01 in Opti-Pro SFM in the presence of 2 µg/ml of trypsin acetylated (Sigma, Chemical Co., St. Louis, MO) at 34°C for 1 h. Then, cells were washed with PBS three times and incubated in Opti-Pro SFM supplemented with 2 µg/ml of trypsin acetylated at 37°C for 24 h. The viral RNA from tissue culture supernatant was mixed with the lysis buffer containing carrier RNA derived from uninfected A549 or MDCK cells and was extracted using MagMAX™-96 Blood RNA Isolation Kit (Ambion, Austin, TX) on King Fisher purification systems (Thermo Scientific, Cambridge, MA). The total RNA from cultured cells was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany) followed by DNase I (Qiagen, Hilden, Germany) treatment.

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Manufacturer protocol

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