Quantitect sybr green kit

The total RNA from the outer canopy leaves was extracted using the MasterPure Plant RNA purification kit by manufacturer instruction (Epicentre, Madison, WI, USA). The integrity of the RNA was tested using 1% TAE agarose gel and was then quantified using a Nanodrop ND-1000 spectrophotometer (Thermo scientific, Waltham, MA, USA). A total of 250–500 ng of high-quality RNA was used to prepare low molecular weight cDNA synthesis using a miSCRIPT Plant RT kit per manufacturer protocol (Qiagen, Venlo, The Netherlands). This prepared cDNA was then used to run qRT-PCR reactions in technical duplicates using Quantitect SYBR green kit according to manufacturer instructions (Qiagen, The Netherlands). The run was performed on a Roter-Gene Q-6000 machine (Qiagen, The Netherlands). U6-snoRNA and 5.8S rRNA were used to normalize miR156 and miR172 transcript abundance. Primers were used to amplify transcripts from Ahsan et al. [79 (link)].

Mice livers were harvested and preserved in the RNAlater solution (Sangon Biotech, China) at −20°C until ribonucleic acid (RNA) extraction. Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA), and complimentary deoxyribonucleic acid was synthesized with SuperScript (TaKaRa Bio, Japan). RT-PCR was carried out using a QuantiTect SYBR Green Kit (Qiagen, Germany) on an ABI Prism 7500 RT-PCR instrument equipped with appropriate software (Applied Biosystems, CA, USA). The oligonucleotide sequences used for RT-PCR were as follows (Sangon Biotech, China): glyceraldehyde 3-phosphate dehydrogenase, sense 5′-GTCTTCACTACCATGGAGAGG-3′ and antisense 5′-TCATGGATGACCTTGGCCAG-3′; p47phox, sense 5′-GTCGTGGAGAAGAGCGAGAG-3′ and antisense 5′-CGCTTTGATGGTTACATACGG-3′; gp91phox, sense 5′-CCGTATTGTGGGAGACTGGA-3′.

Total RNA from infected or transfected HEK-293T cells was extracted using Nucleospin RNA kit (Machery Nagel) according to the manufacturer's protocol and reverse transcribed into cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen, Life Technologies). A SYBR Green-based real-time PCR assay (QuantiTect SYBR Green kit, Qiagen) for relative quantification of transcripts was performed on 384-well plate QuantStudio 6 Flex real-time PCR system (Applied Biosystems) according to protocol as previously described^{19 (link),20 (link)}. The relative quantities were determined by using raw Cp values as input for qBase, a flexible and open source program for qPCR data management and analysis^{48 (link)}; the primers used in this manuscript were listed in Suppl. Table 2.

Samples for RNA analysis were harvested at 4°C by centrifugation, the cell pellet resuspended in Trizol solution and the RNA extracted according to the manufacturer’s instructions (Invitrogen, Grand Island, NY). RNA samples were subsequently treated with TURBO DNase (Ambion, Austin, TX), following the manufacturer’s instructions. Quantitative RT-PCR was performed using a one-step QuantiTect SYBR Green kit (Qiagen) following the manufacturer’s instructions. To confirm gene deletions, ackA and pta transcripts were assayed in WT, ΔackA, Δpta and their complements using primers 21–24 (Table 1). To amplify rpoD, ospC and rpoS transcripts, primers 25–30 were used (Table 1). cDNA was synthesized at 42°C for 30 minutes and denatured at 95°C for 15 minutes. Quantitative RT-PCR was performed under standard conditions on a LightCycler (Roche, Indianapolis, IN). The LightCycler software was used for the analysis of fold changes and rpoD normalization.

Reverse transcription was performed with the QuantiTect-RT kit (Qiagen) using 1 µg of total RNA. Specific mRNA expression levels were measured by quantitative realtime-PCR (qRT-PCR) performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using QuantiTect SYBR Green Kit (Qiagen) in a volume of 15 μl including 2 μl of cDNA. Oligonucleotide sequences of used primers are shown in Supplementary Table S4. Melting curves were collected to ascertain specificity of PCR-products. Changes in mRNA-expression were calculated by the ΔΔ-Ct method and are presented as foldchange in relation to expression of a reference gene (HPRT or Sdha).

RNA was isolated from cells by the Invitek Mini Kit (Invitek) or from tissues using TriZOL (Sigma-Aldrich), followed by clean-up on the Invitek Mini Kit. 1 μg RNA was reverse-transcribed using the iScript Select cDNA Synthesis Kit (Bio-Rad). Quantitative Real-Time PCR was performed using the QuantiTect SYBR Green Kit (QIAGEN). RNA Polymerase II (PolII) expression was used for normalization, while fold change was calculated using 2^−ΔΔCt. Primer sequences are available in Table 1.