The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages
The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages
L. monocytogenes 1/2a EGD-e were cultured in Brain Heart Infusion broth at 37C with shaking at 180 rpm. Eukaryotic cell growth conditions of murine macrophages P388D1 were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories) in 85 mm tissue culture plates for RNA isolation. P388D1 murine macrophages were cultured in 85 mm tissue culture plates to about semi confluency. L. monocytogenes 1/2a EGD-e was infected to the cell monolayer at a MOI of 100 per eukaryotic cell. Infection was carried for 45 min and followed by addition of fresh medium containing 20 ug/ml gentamicin. The medium of the plates (containing 20 ug/ml gentamicin) infected with L. monocytogenes 1/2 EGD-e were replaced after 2 h post infection with fresh medium containing 50 ug/ml gentamicin. At each step the plates were washed extensively with 1x PBS. Incubation of the bacterial tissue culture plates was carried out in a humidified incubator for up to 4 h post infection. For RNA isolation from L. monocytogenes grown extracellularly in BHI, aliquots of 0.5 ml bacterial culture were treated with 1.0 ml RNA protect (Qiagen) for 5 min, the bacterial cells were collected by centrifugation for 10 min (8000 x g) and subsequently stored at -80C until use. RNA extraction from intracellularly grown L. monocytogenes in macrophages was performed as described previously (Chatterjee, 2006) with exeption of a MOI of 100. Briefly, infected host cells were lysed using cold mix of 0.1% (wt/vol) sodium dodecyl sulfate, 1.0% (vol/vol) acidic phenol and 19% (vol/vol) ethanol in water. The bacterial pellets were collected by centrifugation for 3 min (16000 x g).Total RNA was extracted using miRNeasy kit (Qiagen) with some modifications. The collected pellets were washed with SET buffer (50 mM NaCl, 5 mM EDTA and 30 mM Tris-HCl [pH 7.0]) containing 10% SDS. After centrifugation at 16,000 g for 3 min pellets were resuspend into 0.1 ml Tris-HCl [pH 6.5] containing 50 mg/ml lysozyme, 25 U of mutanolysin, 40 U of SUPERase, 0.2 mg of proteinase K (Ambion) and finally treated with 4 U of DNaseI (RNase free) (Ambion) at 37C for 30 min at 350 rpm. 600 ul of QIAzol was added, mixed gently and incubated for 3 min at room temperature. An additional incubation at room temperature was done after adding 0.2 volume chloroform followed by centrifugation at 16,000 g at 4C for 15 min. The upper aqueous phase, containing RNA, was transferred to a new collection tube and 1.5 volumes of 100% ethanol was added and mixed thoroughly. The probes were transferred into columns supplied with the miRNeasy Kit and treated as described in the Qiagen manual, including an on-column DNase digestion (RNase-Free DNase, Qiagen). RNAs were eluted by RNase-free water and stored at -80C until needed. The quantity of the isolated total RNA was determined by absorbance at 260 nm and 280 nm, and the quality was assessed using Nano-chips for Agilents 2100 Bioanalyzer. For detection and estimation of the small RNA fraction within the isolated total RNA, a small RNA-chip (Agilent) was used, which visualize RNAs with sizes ranging from 20 to 200 nucleotides.
Experimental Factor: GROWTHCONDITION
extracellular