TRIzol™ LS Reagent

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TRIzol LS reagent is intended for liquid samples, using solid samples will decrease RNA yield

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TRIzol LS reagent is intended for liquid samples, using solid samples will decrease RNA yield

Publication protocol

For isolation of RNA, Trizol LS reagent (Invitrogen) was used. RNA was treated with DNase (TURBO DNA-free; Ambion). For RNA-free samples RNA was treated with RNase A (Roche, 10 mg/ml, heat-inactivated) for 30 min at 37°C followed by addition of EDTA (pH 8, 2.5 mM) and further incubation for 10 min at 70°C. For control, sample containing only H2O (RNase control) was treated in the same way. mRNA fraction was obtained using the MICROBExpress Kit (Ambion). Briefly, after the Trizol step of the total bacterial RNA isolation fragments shorter than 200 nucleotides were removed from total RNA using miRNeasy Mini Kit (Qiagen). mRNA was then isolated according to the MICROBExpress Kit protocol. Bacterial rRNA was eluted from the MICROBExpress Kit beads by adding TE buffer and incubating for 10 min at 70°C. Bacterial mRNA isolated with the MICROBExpress Kit contained 0.3% bacterial rRNA as assessed with qPCR analysis. Pure bacterial mRNA was obtained from total RNA using the Ribo-Zero rRNA Removal Kit (Epicenter) which yields mRNA without contaminating rRNA, but does not allow elution of rRNA. For DNA preparation, over-night cultures of bacteria were harvested, resuspended in TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA) and treated with lysozyme (Sigma, 2.5 mg/ml) for 30 min at 37°C. Subsequently, RNase A (10 mg/ml), 20% SDS and 0.5 M EDTA were added. After incubation for 40 min at 37°C, 20 mg/ml proteinase K was added, and the samples were further incubated for 30 min at 37°C. DNA was isolated using phenol:chloroform:isoamylalcohol (Sigma-Aldrich). Isolated bacterial DNA was treated with RNase A (10 mg/ml) for 30 min at 37°C. Adherent macrophages and HEK cells were seeded (1x106 cells per 6 cm dish) the day prior to transfection, non-adherent cDCs and THP-1 cells (1x106 cells per 6 cm dish) were seeded 2 hours prior to transfection, PMA-stimulated THP-1 cells were seeded 3 days prior to transfection (1x106 cells per 6 cm dish) with 1 to 5 μg RNA or DNA using DOTAP. The transfection mixture was added to dishes containing 1.5 ml media without antibiotics.

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