Gelstar

Endonucleolytic heteroduplex DNA cleavage analysis was performed using T7-nuclease-I (New England Biolabs, USA) as recommended by the supplier. In brief, heteroduplex and/or perfect match amplicons were incubated with 1 μl T7nuclease in a 20 μl volume at 37°C for 1 h, followed by 3% agarose gel GelStar (Lonza, USA) analyses.

Nondenaturing gel electrophoresis experiments were performed in a 0.5 × TBE buffer in the presence of 20 and 100 mM KCl. Electrophoresis experiments were run at 80 V for 5 h at 4 °C, and the gel was viewed by GelStar (Lonza) staining. RNA samples concentration ranged from 90 to 140 pmol. DNA oligonucleotides dT12 and dT24 were used as molecular markers.

Denaturing gradient gel electrophoresis (DGGE) was carried out as previously reported [18 (link)] using a DCode^TM Universal Mutation Detection System instrument and a Model 475 gradient former according to the manufacturer’s instructions (Bio-Rad Labs, Hercules, CA, USA). The V2-V3 region of the 16S rRNA genes (positions 339 to 539 in the Escherichia coli gene) of bacteria in the gut samples was amplified by primers HDA1-GC and HDA2 as described by Walter et al. [19 (link)]. PCR reaction mixtures and the amplification program were the same as described previously [19 (link)] except that 30 amplification cycles were used. The denaturing gradient was formed using two 8% acrylamide gels (acrylamide-bis 37.5:1) with denaturing gradients ranging from 30 to 70% for analysis of the amplified 16S rRNA fragments. The 100% denaturant solution contained 40% (v/v) deionized formamide, and 7 M urea. PCR products (40 μL) were mixed with 40 μL of loading dye before loading. Gels were run in 0.5 × TAE at 60 °C for 5.2 h at 180 V, 210 mA, stained with Gel Star (Lonza, ME, USA) for 30 min, and analyzed by Chemi Doc MP (Bio-Rad Laboratories, CA, USA). Image Lab software version 5.0 (Bio-Rad) was used for the identification of bands and normalization of band patterns from DGGE gels.

PCR mixtures were prepared in a total volume of 30 μl, consisting of 15 μl of 2× EmeraldAmp MAX PCR master mix (TaKaRa Bio Inc.), 1 μl of each primer (CauF and CauR) at 10 μM, and 2 μl of DNA. PCR was performed in a T100 thermal cycler (Bio-Rad Laboratories, Inc.). The thermal profile included an initial denaturation for 3 min at 95°C followed by 30 cycles of 20 s at 95°C, 20 s at 68°C, and 20 s at 72°C. The presence of amplicons was examined electrophoretically on 2% agarose gels stained with GelStar (Lonza).

Total RNAs were extracted from lungs using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RT-PCR was performed on 10 ng of total RNA using GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit (Roche Life Science, Indianapolis, Indiana). The following primers targeting respectively ADAM28 exon 11 and exon 2 were used: (F) 5′-CTACTTGAGCTGCAAGTGTCC-3′, (R) 5′-CAGGTCCTTGCATCACAGCAT-3′, (F) 5′-GTAAAAGAGAGACCCAAGAGCCAG-3′ and (R) 5′-GTAGTCCTTGACAGGTGCTGATG-3′. RT-PCR products were resolved on 10% acrylamide gels and analyzed with a fluorescence imager (LAS-4000; Fujifilm, Minato-ku, Tokyo) after Gel Star staining (Lonza, Bâle, Switzerland). Gene expression levels were measured as the ratio between expression values of the gene of interest and internal 28S rRNA.

Total LEC RNA was isolated using the High Pure RNA isolation Kit (11828665001, Roche). RT-PCR was performed with 10 ng of total RNA with the GeneAmp® Thermostable rTth Reverse Transcriptase RNA PCR Kit (Applied Biosystems). RT-PCR products were observed after electrophoresis in 10% acrylamide gels and staining with Gel Star (50535, Lonza). The following primers were used: 5′-TGTCAGTCAGCTGGTCCTTG-3′ (reverse primer) and 5′-CCTGGGCATGTATGAGTGTG-3′ (forward primer) for uPARAP; 5′-TGCAGAACTCCACGATCACC-3′ (reverse primer) and 5′-CCCACGCAGACATCAAGACG-3′ (forward primer) for VEGFR-3; 5′-AGCTGCCTGACCACGCAATGT-3′ (reverse primer) and 5′-TTCCACGTGACCAGGGGTCCT-3′ (forward primer) for VEGFR-2; and 5′-GGATTCTGACTTAGAGGCGTTCAGT-3′ (reverse primer) and 5′-GTTCACCCACTAATAGGGAACGTGA-3′ (forward primer) for 28S (internal control).