High capacity archive kit

For both human PBMCs and mouse samples, total RNA was isolated using TRIzol reagent (Life Technologies), and treated with DNase (Ambion/Life Technologies AM1906) after extraction. Only total RNA extracts with an OD260/OD280 ratio above 1.96, indicating relatively pure RNA, were processed for RT-PCR, the remainder undergoing re-extraction [39 (link)]. Total RNA was used to prepare cDNA via the Applied Biosystems High Capacity Archive Kit (#4368814). For detection and measurement of expression, Fermentas Maxima SYBR Green/ROX qPCR Master Mix (#K0222) was used. PCR mixtures were run on a Thermo Scientific PikoReal real-time PCR System using the following conditions: 95ᵒC for 10 minutes, followed by 40 cycles of 95ᵒC for 30 seconds, 60ᵒC for one minute and 72ᵒC for one minute. Cycle threshold (CT) value was used for relative quantification. For human PBMC samples, all values were normalized to three housekeeping genes, GAPDH, TFRC and b-actin using a geometric mean, and run in triplicate [40 ]. Mouse mRNA samples were normalized to b-actin. Primer sequences are listed in S1 Table, with human primers (h) used in PBMC samples and mouse (m) primers used in mouse brain extracts.

Total RNA was isolated from bacteria growing cultures at the exponential phase of growth using the RNeasy midi Kit (Qiagen). Cell lysates were extracted twice with phenol-chloroform before being loaded onto RNeasy midi columns for RNA purification. DNA, potentially contaminating the RNA preparations, was removed by incubation with RNase-free DNAse (Ambion) and its absence was tested by quantitative real time PCR amplification in the absence of reverse transcriptase. Complementary DNA was synthesised using the High Capacity Archive kit (Applied Biosystems). Quantitative real time PCR (qRT-PCR) was performed using SYBR Green technology as previously described [3 (link)]. Three biological samples from the different bacterial cultures were amplified in triplicate in separate PCR reactions. All PCR products were between 50 and 150 bp in length.
A melting curve analysis was conducted after amplification to distinguish the targeted PCR products from the non-targeted ones. The melting curves were obtained by heating at temperatures ranging from 60°C to 95°C at a rate of 0.2°C per sec, with continuous fluorescence scanning. The hrdB transcript was carried out as an internal control to quantify the relative expression of the target genes as before [3 (link)]. The oligonucleotides used as primers were previously described [3 (link)].