Bromo chloro propane

Whole TA muscle was used for RNA isolation. 50 mg cryopreserved TA muscle was homogenised (Potter S 8533024, B. BRAUN) in 700 µL TRI reagent (Invitrogen, 11312940, Carlsbad, CA, USA) and incubated at room temperature (RT) for 5 min. Samples were centrifuged for 10 min (4 °C, 12,000 g). Supernatant was transferred to a new tube and 70 µL bromochloropropane (Sigma-Aldrich, B9673, Saint Louis, MO, USA) was added. Lysates were inverted and incubated at RT for 5 min and centrifuged (4 °C, 12,000 g, 10 min). RNA containing supernatant was transferred to a new centrifuge tube and washed with 100% ethanol 2:1. RNA was further isolated using the RiboPure RNA purification kit (Thermo Fisher Scientific, AM1924, Waltham, MA, USA). Then, 500 ng RNA and 4 µL SuperScript VILO Mastermix (Invitrogen, 12023679, Carlsbad, CA, USA) were diluted to 20 µL in RNAse free water and reverse transcription was performed in a 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA), using the following program: 10 min at 25 °C, 60 min at 42 °C and 5 min at 85 °C. cDNA was diluted 10 × in RNAse-free water.

Isolation of RNA from tissue samples was performed using a modification of the method described by Hansen, et al.[48 (link)]. Up to 200mg of tissue was disrupted in TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) with stainless steel beads (2x5 mm) using a TissueLyser (Qiagen, Germantown, MD) for three minutes at 25 r/s twice. Following homogenization, samples in TRIzol were separated using bromo-chloro-propane (Sigma, St. Louis, MO). The aqueous phase was collected into a new tube and glycogen was added as a carrier. The samples were washed in isopropanol and ethanol-precipitated overnight at -20°C. RNA was then fully re-suspended in 5 mM Tris pH 8.0.

Homogenized mouse spinal cords (L4-spine level) or primary microglia culture were lysed with 1 ml TRIzol Reagent (Invitrogen). Nucleoprotein complexes were then separated through bromochloropropane (Sigma-Aldrich), and the samples were centrifuged at 12,000 rpm at 4˚C for 15 min. Total RNA was precipitated with an equal volume of isopropanol. The samples were incubated at 25˚C for 10 min and centrifuged at 12,000 rpm at 4˚C for 15 min. The RNA pellets were collected and washed with ethanol by vigorous mixing. RNA was obtained by centrifugation at 7,500 rpm at 4˚C for 10 min. After air-drying, the RNA pellets were dissolved in RNase-free double-distilled water. Reverse transcription was performed using a Reverse Transcription Kit (Applied Biosystems) to synthesize cDNA. Quantitative real-time PCR (qPCR) was performed using FastStart Universal SYBR Green Master with ROX (Roche), and cDNA was amplified using a StepOnePlus RealTime PCR System (Applied Biosystems). PCR cycling conditions were set as follows: 95°C for 10 min, then 40 cycles of 95°C for 15 s, 60°C for 1 min, and 72°C for 20 s. The specificity of the SYBR green assay was confirmed by melting-point analysis. Expression data was calculated from the cycle threshold (Ct) value using the ΔCt method for quantification. Primer sequences are listed in Supplementary Table 1.

Tissue samples, cut to 0.5 cm thickness on at least one side, were stored in RNAlater at 4°C for 2–7 days. RNA was recovered from tissue samples using a modification of the method described by Hansen et al., 2013 [65 (link)]. Briefly, up to 200 mg of tissue was disrupted in TRIzol (Lifetechnologies) with 2 x 5 mm stainless steel beads using the TissueLyser (Qiagen) for 3 minutes at 25 r/s twice. Following homogenization, samples in TRIzol were separated using Bromo-chloro-propane (Sigma). The aqueous phase was collected, and glycogen was added as a carrier. The samples were washed in isopropanol and ethanol precipitated. RNA was fully re-suspended in 5 mM tris pH 8.0.

To study the host immune response to sublethally injured C. jejuni, RNA was extracted from spleen, liver, and ileum of four mice in each group on day 3 and day 7 p.i. Total tissue RNA was extracted using TRIzol (Invitrogen, USA). Approximately 30 to 40 mg of tissue previously stored in RNALater was homogenized using an IKA T10 basic homogenizer (Wilmington, NC, USA) in 1 mL TRIzol reagent and 10 μL of β-mercaptoethanol (Merck, Australia). During homogenization, samples were maintained on ice and further incubated for an additional 7 min on ice. Subsequently, 0.2 mL of bromochloropropane (Merck, Australia) was added to each sample. The samples were incubated on ice for 5 min and centrifuged at 12,000 × g for 15 min at 4°C. The aqueous phase of the supernatant was transferred into 0.5 mL chilled (−20°C) isopropanol (Merck, Australia) and incubated for 1 h in a −20°C freezer. The samples were then centrifuged at 12,000 × g for 10 min at 4°C. The extracted RNA was washed in 1 mL of 75% ethanol, centrifuged, subsequently air dried for 5 to 10 min on ice, and resuspended in 50 μL water. The quality and quantity of RNA were measured with a Nanodrop instrument (ThermoFisher, Scientific, Australia) and stored at −80°C.

To study the host immune response to sublethally injured C. jejuni, RNA was extracted from spleen, liver, and ileum of four mice in each group on day 3 and day 7 p.i. Total tissue RNA was extracted using TRIzol (Invitrogen, USA). Approximately 30 to 40 mg of tissue previously stored in RNALater was homogenized using an IKA T10 basic homogenizer (Wilmington, NC, USA) in 1 mL TRIzol reagent and 10 μL of β-mercaptoethanol (Merck, Australia). During homogenization, samples were maintained on ice and further incubated for an additional 7 min on ice. Subsequently, 0.2 mL of bromochloropropane (Merck, Australia) was added to each sample. The samples were incubated on ice for 5 min and centrifuged at 12,000 × g for 15 min at 4°C. The aqueous phase of the supernatant was transferred into 0.5 mL chilled (−20°C) isopropanol (Merck, Australia) and incubated for 1 h in a −20°C freezer. The samples were then centrifuged at 12,000 × g for 10 min at 4°C. The extracted RNA was washed in 1 mL of 75% ethanol, centrifuged, subsequently air dried for 5 to 10 min on ice, and resuspended in 50 μL water. The quality and quantity of RNA were measured with a Nanodrop instrument (ThermoFisher, Scientific, Australia) and stored at −80°C.