Real time rt pcr

For protein analysis in lung tissue, lung biopsy tissue from IPF/UIP patients or from the normal margins of lung tumor resections, or lungs from mice, were homogenized in antiprotease buffer (Roche Diagnostics) and processed as previously described (46 (link)). BAL was taken from the mice by flushing the lungs with sterile PBS, while serum was prepared from a postmortem blood draw. Whole lung lobes were dissected for histological and biochemical analysis. Cytokine levels were measured by bead based LuminexÔ analysis or specific ELISA (R&D Systems). In some instances, mediator levels were normalized to protein content, which was determined using a standard Bradford protein assay. For gene analysis, total RNA was obtained using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Gene expression levels were quantitated using real time RT-PCR (Applied Biosystems), according to the manufacturer’s protocols. Undetectable expression was set as zero for comparisons. Transcript levels of genes of interest were normalized to β-actin or 18S mRNA.

For single cell samples or for lung tissue analysis, lung biopsy tissue from IPF/UIP patients, NSIP patients, COPD patients or the normal margins of lung tumor resections; or lungs from mice were processed for total RNA using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Gene expression levels were quantitated using real time RT-PCR (Applied Biosystems) according to the manufacturer’s protocols. Transcript levels of genes of interest measured by quantitative RT-PCR were normalized to housekeeping gene mRNA. For protein analysis, collagen levels were determined in lung homogenates using an established hydroxyproline assay as previously described^{41 (link)}. Cytokine levels were measured by MSD analysis (Mesoscale), or specific ELISA to mouse TGFβ1 (R&D Systems), or p21 according to manufacturer’s protocols.

Gene expression levels were quantitated using real time RT-PCR (Applied Biosystems) according to the manufacturer's protocols. Transcript levels of genes of interest measured by quantitative RT-PCR were normalized to housekeeping gene mRNA. For protein analysis, collagen levels were determined in lung homogenates using an established hydroxyproline assay as previously described^{21 (link)}. TNFα protein levels were measured by MSD analysis (Mesoscale Discovery), according to manufacturer’s protocols.

Total RNA was extracted from colonic mucosa using an RNeasy plus mini kit (Qiagen GmbH, Hilden, Germany) and from RAW264.7 cells using TRIzol Reagent. We purified RNA using the lithium chloride procedure described previously,^{(25 (link))} since DSS is known to interfere with PCR reactions. The cDNA was synthesized using a reverse transcriptase kit (Invitrogen) with an oligo-dT primer. After cDNA synthesis, real-time RT-PCR (Applied Biosystems, Carlsbad, CA) was performed using SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA). The amplification programs were set as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. The primer sequences are shown in Table 2. All sample mRNA levels were normalized to GAPDH for animal or β-actin for RAW264.7 cells and the relative mRNA levels were calculated.

RNA was isolated from leaves, roots, or nodules from three-pooled plants (from independent experiments each) following the protocol previously described by Abreu et al. (2017) (link). Briefly, RNA was extracted using Tri-Reagent^® (Life Technologies, Carlsbad, CA) followed by a DNase treatment and later cleaned with RNeasy Minikit (Qiagen, Valencia, CA). Denaturing agarose gel was used to verify RNA quality. One microgram of DNA-free RNA was employed to generate cDNA by using PrimeScript RT Reagent Kit (Takara). Gene expression was determined by quantitative Real time RT-PCR (9700, Applied Biosystems, Carlsbad, CA, United States) using primers listed in Supplementary Table S1. The M. truncatula ubiquitin carboxyl-terminal hydrolase gene was used to normalize the results. Real-time cycler conditions have been previously described (González-Guerrero et al., 2010 (link)). The threshold cycle (Ct) was determined in triplicate. The relative levels of transcription were calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)). As control, a non-RT sample was used to detect any possible DNA contamination.

Nasal or nasopharyngeal swabs were collected from patients <14 years of age, whereas from patients aged >14 years, throat and nasal swabs were collected in viral transport medium (Hi-media) and were processed immediately (within 3–4 h) at the Influenza laboratory of Sher-i-Kashmir Institute of Medical Sciences. Samples were analyzed for diagnostic purposes using real-time RT PCR (Applied Biosystems) employing the CDC protocol [23 ] for detection of influenza. Influenza A-positive samples were further subtyped using primers and probes for A/H1N1pdm09 and A/H3, and influenza B positive samples were subtyped into B/Yamagata and B/Victoria lineages. Haemagglutinin (HA) sequencing and phylogenetic analysis was carried out using standard assay procedures at the Cnr Virus Des Infections Respiratoires France Sud, Lyon, France, and the nucleotide sequences were submitted to the GenBank database under accession numbers EPI 180439, EPI 1804279, EPI 1804213, EPI 1804207, EPI 1804169, EPI 1804219, EPI 1804291, EPI 1804301, EPI 1804145, EPI 1804299, EPI 1804297, EPI 18004189, and EPI 1804155 for influenza B (Victoria lineage). Representative A/H1N1 nucleotide sequences were submitted to the Genbank database under accession numbers EPI 1804129 and EPI 180435, whereas the A/H3N2 sequences were submitted to the database under the accession numbers EPI 1803979 and EPI 1696367.