Faststart universal sybr green master kit

The specific primers for SERPINC1 amplification were: Forward 5′–gttgc ccttc aaagg tgatg a-3′, Reverse 5′-cagat cgaca aggcc catgt c-3′. Total RNA was extracted from cells with TRIzol^® reagent (Invitrogen, Carlsbad, CA). Then, the RNA integrity was confirmed by electrophoresis and the concentration was determined. RNA was reverse-transcribed with Superscript II (Invitrogen) and random hexamer primer, and quantitative PCR was performed with a FastStart Universal SYBR Green Master Kit (Roche) and an ABI PRISM 7900HT system (Applied Biosystems). The reaction protocol was set as 95 °C for 5 min and 35 cycles of 95°C for 15 s, 60°C for 60 s, and 72°C for 5 min. The expression of genes of interest was normalized to that of the reference gene (β-actin), and expression was calculated with the 2^−ΔΔCt method.

Owing to the hemizygous state of the deletion in females, the qPCR analysis was performed using a FastStart Universal SYBR Green Master kit (Roche Applied Science, Germany) and on ABI 7900-HT system. An unrelated female control was also included to confirm successful amplification. The PCR mix was preheated at 94 °C for 5 min and then amplified in 40 cycles of 94 °C for 20 s and 60 °C for 15 s. Each PCR reaction was run in triplicate. To verify the specificity of PCR products, melting curve analysis was performed at the end of each PCR reaction. GAPDH was used as internal control. The sequences of all primers used are listed in Table 1. Data analysis was performed using the 2^-△△Ct method as previously described [13 (link)].

Total RNA was extracted from lung tissue using Trizol reagent (Invitrogen Life Technologies) according to the manufacturer's protocol. Total RNA samples were reverse-transcribed to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qPCR was performed using 1 μL of cDNA and a FastStart Universal SYBR Green Master kit (Roche). The following primer sets were used: β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′; TGF-β1, 5′-GCGGACTACTATGCTAAAGATGT-3′ and 5′-GTTGTGTTGGTTGTAGAGGGGCA-3′; RANTES, 5′-GTACATCACCATGGCGTATG-3′ and 5′-TCTTCTCTGGGTTGGCACACA-3′; eotaxin, 5′-CCATCTGTCTCCCTCCACCATG-3′ and 5′-ATCCCACATCTCCTTTCATGCC-3′; GATA-3, 5′-GAAGGCATCCAGACCCGAAAC-3′ and 5′-ACCCATGGCGGTGACCATGC-3′; IL-6, 5′-CTGGTGACAACCACGGCCTTCCCTA-3 and 5-ATGCTTAGGCATAACGCACTAGGTA-3′; IL-13, 5′-CTGCAGTCCTGGCTCTCG-3′ and 5′-CTTTTCCGCTATGGCCACTG-3′; IL-17, 5′-AGATCTGGACGCGCAAACATGAG-3′ and 5′-GGGTCGTCGACGGGTCTCTGTTTAG-3′.

Streptomyces sp. 139, mutant strain D2 and the complemented strain C2 were cultured at 28 ℃ for 48 h in TSB. 10 mL of the culture were inoculated into 50 mL fresh TSB and were cultured at 28 ℃ for 24 h, 48 h, and 96 h, respectively. At each time point, the mycelia of the three strains were collected by centrifugation (3,000 g; 10 min) and washed with PBS (NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 10 mM, KH₂PO₄ 2 mM, pH 7.4), respectively. Total RNA of each mycelia sample was isolated with the TRIzol System (TransGen) according to the manufacturer’s instructions. Remaining DNA was removed by DNAse (TaKaRa). cDNA was synthesized with Superscript III First-Strain synthesis system kit for RT-PCR (TransGen) according to the manufacturer’s instruction. Primers for each gene are listed in Table 1. 50 ng cDNA was used for each qPCR reaction with FastStart Universal SYBR Green Master kit (Roche) following the manufacturer’s instructions. All transcripts were normalized according to hrdB (RNA polymerase principal sigma factor) transcript quantities.