Light cycler data analysis software

Samples (tissues and blood) were collected from euthanized guinea pigs to determine the viral load as previously described. For tissue DNA extraction, FastPrep 24 (MP Biomedical, Irvine, CA, USA) was used to homogenize tissues as a 10% weight/volume homogenate in Lysing Matrix D (MP Biomedicals). Whole blood was collected into ACD anti-coagulant tubes. DNA was extracted using the QIAcube HT (Qiagen, Germantown, MD, USA) according to manufacturer’s tissue or whole blood protocols. Viral load was determined by real-time PCR on a LightCycler 480 (Roche Applied Science) using GPCMV GP44 primers and hydrolysis probe as previously described [25 (link),26 (link)]. Data were collected by “single” acquisition during the extension step and with the LightCycler Data Analysis Software (Version 1.5.1; Roche Life Sciences, Santa Clara, CA, USA). Standard curve was generated using serial dilutions of GPCMV GP44 plasmid [78 (link)] at known concentrations for quantification and assay sensitivity. The sensitivity of the assay was determined to be 5 copies/reaction. Viral load was expressed as genome copies/mg tissue or ml of blood. Results calculated were a mean value of triplicate PCR runs per sample.

Quantitative polymerase chain reaction (qPCR) was performed using the LightCycler FastStart DNA Master SYBR Green PLUS I kit (Roche) and a LightCycler (Roche), following the manufacturer’s protocol as described previously [34 (link)]. Primers for target IFN stimulated genes (ISGs; Table 1) were synthesized by ACGT Corporation. Standard curves for each gene were generated using cDNAs from uninfected A549 cells and MEFs treated with 1 × 10³ U/mL of human IFN-β-1a and 1 × 10³ U/mL of murine IFN-β1, respectively. qPCR data were analyzed using LightCycler Data Analysis Software (Roche).

RNA was isolated from snap frozen colonic ex vivo biopsies, reverse transcribed and analyzed as described previously [20 (link)]. Murine IL-23p19, IL-22, and IL-18 mRNA expressions were detected by real-time polymerase chain reaction (PCR) with specific primers and quantified by analysis with the light cycler data analysis software (Roche). The mRNA of the housekeeping gene for hypoxanthine-phosphoribosyltransferase (HPRT) was used as reference, the mRNA expression levels of the individual genes were normalized to the lowest measured value and expressed as fold expression (arbitrary units).

Maternal blood was obtained on days 7 and 14 post-challenge, with SG-adapted virus, and analyzed for viral load by qPCR, as described previously [42 (link)]. Briefly, DNA was extracted from either 100 μl citrated maternal blood, or from pup tissues, using 0.05 g of homogenized frozen samples of liver, lung or spleen (QIAamp 96 DNA QIAcube HT Kit, Qiagen, Hilden, Germany). Amplification primers GP83TM_F1 (5’-CGTCCTCCTGTCGGTCAAAC-3’) and GP83TM_R1 (5’-CTCCGCCTTGAACACCTGAA-3’) were used at a final concentration of 0.4 µM, while the GP83 hydrolysis probe (FAM-CGCCTGCATGACTCACGTCGA-BHQ1) was used at 0.1 µM. PCR was performed as previously described [42 (link)], and data were analyzed with the LightCycler Data Analysis Software (version 1.5; Roche), using standard curves generated from known copy numbers of a modified plasmid pCR 2.1 containing GP83 sequences. DNAemia was expressed as the total number of genome copies per mL of blood (limit of detection ~200 copies/mL). For the purpose of statistical comparison, a level of 100 copies/mL was assigned to negative samples. Tissue viral loads were expressed as genome copies per mg of tissue. The limit of detection was approximately two genome copies/mg tissue. For the purpose of statistical comparisons, a value of one copy/mg was assigned to negative samples.

Expression levels of pro- and anti-inflammatory cytokines including IFN-γ, IL-22, IL-17A, and IL-10 mRNA were determined in snap frozen ileal and colonic ex vivo biopsies using Light Cycler Data Analysis Software (Roche) as stated elsewhere (30 (link)). The mRNA of the housekeeping gene for hypoxanthine-phosphoribosyltransferase was used as reference; the mRNA expression levels of the individual genes were normalized to the lowest measured value and expressed as fold expression (arbitrary units) (31 (link)).
For molecular analysis of the intestinal microbiota, DNA was extracted from fecal samples as described previously (24 (link)). Briefly, DNA extracts and plasmids were quantified using Quant-iT PicoGreen reagent (Invitrogen, Paisley, UK) and adjusted to 1 ng/µl. Then, abundance of the main bacterial groups of murine intestinal microbiota was assessed by quantitative real time-PCR with group-specific 16S rRNA gene primers (Tib MolBiol, Berlin, Germany) as described previously (5 (link), 32 (link), 33 (link)). The number of 16S rRNA gene copies per microgram DNA of each sample was determined, and frequencies of respective bacterial groups calculated proportionally to the eubacterial (V3) amplicon.