Quantitative Real-Time Polymerase Chain Reaction

Total RNA was isolated from C. difficile 630Δerm strain grown in TY medium either after 4 h, 6 h or 10 h of growth and from R20291 strain during late exponential growth phase (6 h) as previously described [22] (link). Starvation conditions correspond to a 1 h incubation of exponentially grown cells (6 h of growth) in PBS buffer at 37°C. Strains carrying pRPF185 derivatives were grown in TY medium in the presence of 250–500 ng/mL ATc and 7.5 µg/mL Tm for 7.5 h followed by RNA isolation. The cDNA synthesis by reverse transcription and quantitative real-time PCR analysis was performed as previously described [45] (link). In each sample, the relative expression for a gene was calculated relatively to the 16S gene [81] (link). The relative change in gene expression was recorded as the ratio of normalized target concentrations (ΔΔCt) [82] (link). Strand-specific RT-PCR analysis was performed as previously described [27] (link).
For Northern blot analysis, 5 µg of total RNA was separated on a denaturing 6% or 8% polyacrylamide gel containing 8 M urea, and transferred to Hybond-N⁺ membrane (Amersham) by electroblotting using the Trans-blot cell from Bio-Rad in 1× TBE buffer (89 mM Tris-base, 89 mM boric acid and 2 mM EDTA). Following UV-cross-linking of the samples to the membrane, prehybridization was carried out for 2 h at 42°C in 7 mL of prehybridization buffer ULTRAHyb (Ambion). Hybridization was performed overnight at 42°C in the same buffer in the presence of a [gamma-³²P]-labeled DNA oligonucleotide probe. Alternatively, the probe was synthesized using PCR with 5′end-labeled primer complementary to RNA sequence. After hybridization, membranes were washed twice for 5 min in 50 mL 2× SSC (300 mM sodium chloride and 30 mM sodium citrate) 0.1% sodium dodecyl sulphate (SDS) buffer and twice for 15 min in 50 mL 0.1× SSC 0.1% SDS buffer. Radioactive signal was detected with a Typhoon system (Amersham). The size of the transcripts was estimated by comparison with RNA molecular weight standards (Invitrogen).

Quantitative Real-time PCR (qRT-PCR) was performed on an ABI 7500 real-time system. The cDNA of each sample representing one biological replicate was diluted to a working concentration of 17 ng/µl for the qRT-PCR analysis. The melt temperature was 60°C and product contained between 80 and 200 base pairs (Table 1). The 25 µl reaction system contained 1 µl of diluted cDNA, 11.25 µl of SYBR® Green Real-time PCR Master Mix (TIANGEN, Corp, Beijing, China), and 0.5 µl of each primer. The cycling parameters were as follows: 95°C for 3 min followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 35 s. A 3-fold serial dilution of cDNA was used to construct a standard curve to determine the PCR efficiency that would be used to convert the quantification cycles (Ct-values) into the relative quantities (relative gene expressions).

Three days after MCAO, mice were anesthetized and transcardially perfused with cold PBS. For histologic analysis, each whole brain was delicately harvested from the skull and incubated in 4% paraformaldehyde overnight at 4 °C. Following sequential dehydration in 15% and 30% sucrose, brains were embedded in Tissue-Tek® O.C.T. compound (Sakura® Finetek Inc., USA), frozen in liquid nitrogen, and stored at − 80 °C for immunofluorescence staining. After the cerebellum and olfactory bulbs were removed, the ischemic/ipsilateral hemispheres were collected, frozen and stored in liquid nitrogen until protein was extracted for western blot analysis. For quantitative real-time PCR analysis (qRT-PCR), ischemic/ipsilateral hemispheres snap-frozen in liquid nitrogen were sufficiently lysed with TRIzol Reagent (Invitrogen, USA) and stored at − 80 °C thereafter.

cDNA was generated from mRNA using the High-Capacity RNA-to-cDNA™ Kit (#4388950, Thermo Fisher Scientific, Waltham, Massachusetts, U.S.) following the manufacturer’s protocol. In each reaction, 0.3 µg of RNA was used unless the RNA concentration was too low. In this case, 0.2 µg (1 sample) or 0.1 µg (4 samples), was used depending on the concentration. Our analyses showed that the cDNA and following quantitative real-time PCR (qRT-PCR) reactions were quantitative within a range of 0.1 to 1 μg (linear regression analyses yielded R² values > 0.99 two independent experiments using 1, 0.6, 0.3, 0.2, 0.1 and 0.075 μg of RNA input, data not shown). After completion of the reaction, the cDNA was either stored at 4 °C for less than a week or was immediately used for qRT-PCR analysis. After a sample had an acceptable qRT-PCR result or the cDNA had been stored at 4 °C for a week, the sample was not analyzed again. If the sample's Ct value was above the last standard point’s Ct value and the RNA concentration was high enough, 0.6 µg (6 samples) or 1 µg (5 samples) of RNA was used for the qRT-PCR assay and FMR1 mRNA values were adjusted to reflect the content in 0.3 μg (Fig. 2).