High capacity cdna reverse transcript kit

To verify miRNA sequencing results, miRNA expression was assayed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using the Taqman MiRNA Reverse Transcript Kit (Cat# 4366596, Thermo Fisher, Waltham, MA, USA). SYBR Green-based qRT-PCR was used to measure the relative expression of selected target genes. Five hundred nanograms of the total RNA were reverse transcribed to generate complementary DNA (cDNA) employing the High Capacity cDNA Reverse Transcript Kit (Thermo Scientific, Rockford, IL, USA). The QuantStudio™ 3 Real-Time PCR System (ABI) was used for these assays. Relative expression was calculated using the 2−_ΔΔCT method [23 (link)].

Total hepatic RNA was extracted with TRIzol^® (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA with the High-Capacity cDNA Reverse Transcript Kit (Applied Biosystems) and diluted 1:10 with DNase and RNase-free H₂O. Real-time PCR was performed on an ABI 7900 using the SYBR Green assay (primer sequences were available on request) as described previously [4 (link)]. The delta-Ct method was used to calculate mRNA expression levels (expressed by ratio = 2^-deltaCt *100%), using Cyclophilin A as the control gene.

Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Reverse transcription reactions were performed with total RNA ≥20 ng/μl using the High Capacity cDNA Reverse Transcript Kit (Applied Biosystems, Life Technologies, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions. Real-time PCR (Lightcycler 480II; Roche, Basel, Switzerland) was performed with 2 μl single-stranded cDNA sample with SYBR Green PCR master mix (Applied Biosystems). The sequences of primers used are shown in Additional file 5. The annealing temperature was 60 °C. Each amplification reaction was checked to confirm the absence of nonspecific PCR product by melting curve analysis. The relative gene expression level was calculated and presented with the 2^–ΔΔCt method. Beta-2 microglobulin (β₂M) was used as an endogenous control to normalise specific gene expression in each sample.

Total mRNA for qRT-PCR and PCR array studies was extracted from cells using RNeasy Mini Plus kit (Qiagen, 74134) according to the manufacturer’s protocol. cDNA was synthesized from 1μg of total mRNA using High-capacity cDNA reverse transcript kit (Applied Biosystems, 4368814) following the manufacturer’s protocol. qRT-PCR experiments were performed in a MyiQ thermal cycler (BioRad) with Sybr-Green mastermix (BioRad, 170–8882) and 200 ng of template/reaction using primers (Operon) designed using the Primer3/NCBI Primer-BLAST tool69 (link) (Table 3).
The following thermal cycling settings were used: 95 °C for 10 min. followed by 40 cycles at 95 °C for 30 sec., 62 °C for 1 min. and 72 °C for 1 min. The fold-change values were calculated as delta-delta Ct (ddCt) values from a minimum of three independent biological replicates.
The PCR array study was performed with an 84-gene EMT pathway-specific array (SABiosciences, PAHS-090Z) according to the manufacturer’s protocol. The top 20 genes were ranked by fold change magnitude and analyzed for KEGG pathway enrichment using the DAVID bioinformatics tool70 (link).

An overnight culture of P. aeruginosa was diluted in CAMHB to 1 × 10⁶ CFU/mL, and a 50 µL aliquot was inoculated into a 96-well plate. Fifty microliters of LL-37 or RP557 at half MIC was added to each well. After incubation for 24 h, total RNA was extracted using the RNeasy Protect Bacteria Mini Kit (Qiagen, Hilden, Germany), and quality was assessed by the ratio of A260/A280 and A260/A230. The complementary DNA was synthesized using a High-Capacity cDNA Reverse Transcript Kit (Applied Biosystems, Lithuania, UAB). All samples were stored at −80°C until further analysis. The expression level of genes related to biofilm formation was assessed by qPCR using Rotor-Gene Q (Qiagen, Hilden, Germany). The 20 µL reaction system consisted of 10 µL EzAmp qPCR 2× master mix (SYBR Green, Low Rox, Daejeon, South Korea), 2 µg of cDNA, 10 pmol of forward primer, 10 pmol of reverse primer, and ultrapure water (MilliQ, Darmstadt, Germany) to a total volume of 20 µL. The cycling conditions of qPCR were as follows: initial denaturation at 94°C for 3 min, followed by 40 cycles of 15 s at 94°C, 25 s at 56°C, and 30 s at 72°C. The melting curve was obtained from 60°C to 95°C. The housekeeping gene rpoD was used for normalization. The relative changes in expression level were calculated using the 2^-△△ct method. The primers used in this study are listed in Table 1.

The proinflammatory effects of extracellular mitochondrial components were tested by adding isolated EVs (EVs from 10 × 10⁶ cells) or whole mitochondria (12 µg, except where indicated) to 0.2 × 10⁶ RAW264.7 cells for 16 h. Media and cells were then collected. The presence of IP10 in the media was then determined by ELISA using the Mouse CXCL10/IP-10/CRG2 DuoSet ELISA from the R&D system (cat. DY466) according to the manufacturer’s instructions. The presence of IL-6 in the media was then determined by ELISA using the Mouse IL-6 OptEIA^TM ELISA set from BD biosciences (cat. 555240) according to the manufacturer’s instructions. For mRNA quantification, cellular RNA was extracted using the GENEzolTM TriRNA pure kit (Geneaid, Cat No. GZX100), from which cDNAs were generated using a high-capacity cDNA reverse transcript kit (Applied biosystems, Cat No. LS4368814). qPCRs were done using the Sensifast SYBR No-ROX kit (Bioline Cat no. BIO-98005) for the following genes (primer sequences in Supplementary Table 1): Rsad2, mIfit1, and actin as a loading control.