Ultrahyb solution

Total RNA was extracted from primary rat microglia treated with LPS for 24 h using Trizol reagent (Invitrogen) and run on a 15% Tris/Borate/EDTA (90 mM Tris/64.6 mM boric acid/2.5 mM EDTA (pH8.3)) Urea gel (Invitrogen) and transferred to a Nytran nylon transfer membrane (Ambion, Austin, TX). The membranes were cross linked and prehybridized for at least 1h with Ultra-Hyb solution (Ambion), followed by hybridization to the LNA DIG-probe for miRNA-9 (Exiqon, Vedbaek, Denmark) or the snRNA RNU6B probe according to the manufacturer’s instructions.

Mouse embryonic fibroblasts (MEF) genomic DNA was digested with EcoRI and analyzed by Southern blotting. The genomic blot was hybridized with ³²P random primed probe (Exon 1 region, Figure S1; Table S2) in Ultrahyb solution (Ambion) at 42°C overnight. The blot was washed with 0.3% SSC + 0.1% SDS at 60°C for 1 h followed by 0.1% SSC + 0.1% SDS at room temperature for 1 h and exposed to X-ray film at −80°C.

Bacteria were grown as described for total protein extraction (see above). Cultures were centrifuged for 3 min at 4,400 g and 4°C. Pellets were then frozen in liquid nitrogen and stored at –80°C ON. RNA extractions were performed as previously described (50 (link)). An appropriate amount of RNA was mixed with formaldehyde loading dye (Ambion), denatured for 5 min at 65°C and run in 1.25% agarose gels. Gels were submerged in an ethidium bromide solution and RNA integrity and loading were verified by exposure to UV light. RNAs were then transferred to 0.2 μm pore size Nitran N membranes (GE Healthcare Life Sciences) by capillarity, using NorthernMax Transfer Buffer (Ambion), for 1.5 h at RT. The transferred RNAs were crosslinked to the membranes by exposing them to UV light inside a UV Stratalinker 1800 (Stratagene). Membranes were then placed into hybridization tubes and prehybridized with ULTRAHyb solution (Ambion) for at least 30 min at 40°C (for oligonucleotide probe) inside a rotating oven. After the prehybridization step, the corresponding radioactively labeled oligonucleotide was added and incubated ON. Membranes were washed three times for 5 min by addition of preheated 2X SSC, 0.1% SDS at 40°C followed by several washes with 0.2X SSC, 0.1% SDS at RT until background signal was eliminated. Membranes were developed by autoradiography for different time periods.

For northern blot analysis of RyhB RNA, 5 µg of total RNA from each strain was resolved on a Bio-Rad Criterion 10% Tris-borate-EDTA (TBE)–urea polyacrylamide gel in 1× TBE buffer at 100 V for 1 h. The resolved RNA samples were then transferred to a Zeta-probe GT membrane (Bio-Rad) by wet electroblotting at 200 mA for 2 h in 0.5× TBE and cross-linked to the membrane by UV irradiation. The resulting membrane was hybridized with the biotinylated ryhB and ssrA probes in ULTRAhyb solution (Ambion) overnight at 42°C and further incubated with a streptavidin-conjugated alkaline phosphatase. The blot was then developed using the BrightStar BioDetect kit (Ambion) according to the manufacturer's instructions. Northern blots were imaged by capturing the chemifluorescence using the Bio-Rad ChemiDoc MP imager.