Zr duet dna rna miniprep kit

RNA templates were extracted by a ZR-Duet DNA/RNA MiniPrep kit (Zymo Research). The expression levels of three different genes encoding resistance–nodulation–division (RND) family pumps (adeB, adeG and adeJ) and two different genes encoding outer-membrane porins (oprD and carO) were analysed by qRT-PCR using a StepOnePlus Real-Time PCR System (Applied Biosystems) (Peleg et al., 2008 (link); Fernando & Kumar, 2012 (link); Zander et al., 2013 (link)). The primers used for the analysis are listed in Table 1. The housekeeping gene 16S rRNA was used as a control (Coyne et al., 2010 (link); Srinivasan et al., 2011 (link); Hou et al., 2012 (link)). Reactions (20 µl) were set up using 400 nM primers and 2 µl cDNA template (diluted 1 : 10) with SYBR Premix Ex Taq II (Tli RNaseH Plus) and ROX plus (Takara Bio). The data analysis was carried out using StepOne software. The expression of each target gene was normalized based on the level of the 16S rRNA mRNA gene and was expressed as a relative rate compared with that in the susceptible isolate of each pair (the expression of A. baumannii Tokai strain 9 was taken as 1.0). Experiments were conducted at least three times independently and all reactions were carried out in triplicate.

Tumor and adjacent tissue samples including two colon samples and one stomach sample were collected and RNA was isolated using the ZR-Duet DNA/RNA Miniprep Kit (Zymo Research). Total isolated RNA was used to construct the library with the NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacture's protocol. Sequencing reactions were executed on the NextSeq 500 platform using paired-end mode, yielding at least 32 M reads per sample.

Reverse transcription-quantitative PCR (RT-qPCR) was performed to determine the level of mRNA expressed in NT and HIF1α KO cells. RNA was isolated and purified from cells using a ZR-Duet DNA/RNA miniprep kit (Zymo Research). Next, 1 μg of RNA was converted to cDNA using an oligo(dT) primer and the Transcriptor first-strand cDNA synthesis kit (Roche Molecular Systems). qPCR was performed using PowerUp SYBR green (Applied Biosystems Thermo Fisher Scientific) on an ABI real-time qPCR 7300 instrument. H6PD was used to normalize all samples. The following primer pairs were used: IDO1 pair 1 (5′-GGCTTTGCTCTGCCAAATCC-3′ and 5′-TTCTCAACTCTTTCTCGAAGCTG-3′), IDO1 pair 2 (5′-GCATTTTTCAGTGTTCTTCGCATA-3′ and 5′-CATACACCAGACCGTCTGATAGCT-3′), VEGF (5′-TCCTCACACCATTGAAACCA-3′ and 5′-GATCCTGCCCTGTCTCTCTG-3′), and H6PD (5′-GGACCATTACTTAGGCAAGCA and 5′-CACGGTCTCTTTCATGATGATCT-3′).

Total RNA was extracted at least twenty-four hours after CAP treatment from cells using the ZR-Duet DNA/RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. To quantify the level of mature miR-19a, reverse transcription with the extracted RNA was carried out using the miScript II RT Kit (Qiagen, Valencia, CA, USA). Quantitative RT-PCR was then conducted with the miScript SYBR Green PCR Kit (Qiagen) and miScript Primer Assays as the primers. In the case of the quantification of protein-coding genes’ expression level, cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Japan), and KAPA SYBR FAST qPCR Kit Master Mix ABI Prism (Kapa Biosystems, Inc., Wilmington, MA, USA) was used for real-time PCR reaction. U6 and GAPDH were used for normalizing the expression of miR-19a and the protein-coding genes, respectively, with the 2^−ΔΔCt method. Each RT-PCR reaction was assayed in triplicate on an ABI 7300 instrument (Applied Biosystems, Foster City, CA, USA). The primers used for the amplification of miRs and selected genes are listed in Supplementary Table S2.

Total RNA was prepared from the cultured cells using the ZR-Duet DNA/RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) and reverse-transcribed to cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). The cDNA was amplified using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, USA) with an ABI 7300 instrument (Applied Biosystems, Foster City, CA, USA). The relative gene expression was calculated using the 2^–∆∆Ct method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The PCR condition was as follows: Enzyme activation at 95 °C for 3 min; 40 cycles of denaturation at 95 °C for 3 s, annealing/extension at 60 °C for 40 s.