RNA from mCCD cells was extracted using a RNeasy mini kit (Qiagen). cDNA was synthesized with PrimeScript RT reagent Kit (Takara) at 37 °C 15 min, 85 °C 5 s, and 4 °C. Quantitative real-time (qRT)-PCR was performed using PowerUp SYBR Green Master Mix (Thermo Fisher scientific) by incubating 2 min at 50 °C, 2 min at 95 °C; followed by 40 cycles of: 95 °C 15 s, 60 °C 1 min before being held at 4 °C. Primers targeting MASP2 (5′-ACCGCTGCGAGTATGACTTT-3′, 5′-TGCATAGAAGGCCTCAAACC-3′) were applied. The primers does not target the splice varian MAp19. The data were analyzed by 7500 Fast Real-Time PCR systems with software v.2.04 (Applied Biosystems, Foster City, CA, USA). Human kidney and liver -tissue and mice tissue were RNA-extracted by phase separation using Trizol reagent (Merck). Turbo-DNAse free kit (Invitrogen) was applied, and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). PCR amplification was performed with predesigned primer H_MASP2_3 (Sigma-Aldrich, RefSeq ID NM_006610) using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) at 95 °C for 3 min and 36 cycles of 95 °C 20 s, 60 °C 30 s, 72 °C 30 s, and 72 °C for 10 min before being held at 4 °C. The samples were separated on a 2% agarose gel with loading dye and a size marker (Sigma-Aldrich) at 125 V. H2O, and samples without reverse transcriptase, GADPH, and β-actin were included as controls.
Powerup sybr green master mix
Manufactured by Thermo Fisher Scientific
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PowerUp SYBR Green Master Mix is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green I dye, Dual-Lock DNA polymerase, and necessary reagents for efficient and sensitive quantification of DNA targets.
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3 763 protocols using powerup sybr green master mix
1
Quantitative Real-Time PCR Analysis of MASP2
2
Quantitative PCR Transcript Profiling
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Complementary DNA (cDNA) synthesis was carried out using DNA-free RNA with the RT First Strand Kit according to the manufacturer’s instructions (Qiagen). Each cDNA sample was used at 120 ng per well in 96-well plates. qPCR was conducted using the PowerUP SYBR Green Master Mix (Thermo Fisher Scientific, USA), with each reaction having 5 μl of PowerUP SYBR Green Master Mix, 2 μl of 1 μM forward and reverse gene-specific primers (table S4), and 3 μl of diluted cDNA experimental sample in a final reaction volume of 10 μl. Reactions were run with the following cycle profile: initial denaturation, 95°C 10 min; 40-cycle amplification/elongation, 95°C 15 s followed by 60°C 1 min. gDNA contamination from each sample was assessed using a total of 120 ng of RNA (no reverse transcriptase) with reaction conditions as listed above, with all samples having CT values of >35. No-template controls were run with each qPCR plate and had CT values of >38. All qPCRs were run and analyzed on the QuantStudio 3 Real-Time PCR System with analysis software v1.5.1 (Applied Biosystems). Each time point was collected in biological triplicates, with qPCRs from each experimental sample done in technical triplicates.
3
Quantifying Gene Expression Using qPCR
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The relative mRNA expression levels were assessed through qPCR with the use of the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) according to the provided manual. In brief, the total RNA from the cell samples was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The quantity and quality of RNA samples were verified using BioAnalyzer 2100 (Agilent, Santa Clara, CA, USA). Next, total RNA (1 μg) underwent reverse transcription using the SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, Waltham, MA, USA). Then, real-time PCR was conducted using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The relative levels of expression were calculated using the 2−ΔΔCt method with GAPDH as the internal control. The primers used in the present study were as follows: WFDC2, sense: 5′-GCT GGC CTC CTA CTA GGG TT-3′, anti-sense: 5′-AAC ACA CAG TCC GTA ATT GGT-3′; HIF1α, sense: 5′-CTC AAA GTC GGA CAG CCT CA-3′, anti-sense: 5′-CCC TGC AGT AGG TTT CTG CT-3′; GAPDH, sense: 5′-GGA GCG AGA TCC CTC CAA AAT-3′, anti-sense: 5′-GGC TGT TGT CAT ACT TCT CAT GG-3′.
4
Quantifying DNA Damage and Exogenous Persistence
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Cells were harvested and washed once with PBS. Genomic DNA extraction was performed using Mag‐Bind Blood & Tissue DNA HDQ kits (Omega Bio‐Tek, Norcross, GA, USA) following the manufacturer’s instructions.
For DSB detection, qPCR primers (TableS1 ) were designed to amplify the genomic sequence containing either the TALEN target sites or upstream (or downstream) of the TALEN target sites as controls. The qPCR was set up with the PowerUp SYBR Green Master Mix (Thermo Fisher) and analyzed on Bio‐Rad CFX (Bio‐Rad, Hercules, CA, USA). qPCR annealing temperature was 60 °C for all primers.
To determine the exogenous dsDNA half‐life, qPCR primers were designed to specifically amplify the CAR sequence. Primer pairs amplifying part of the genomic DNA of actin were used as loading control (TableS1 ). The qPCR was set up with the PowerUp SYBR Green Master Mix (Thermo Fisher) and analyzed on Bio‐Rad CFX. qPCR annealing temperature was 60 °C for all primers.
For DSB detection, qPCR primers (Table
To determine the exogenous dsDNA half‐life, qPCR primers were designed to specifically amplify the CAR sequence. Primer pairs amplifying part of the genomic DNA of actin were used as loading control (Table
5
Real-time PCR Gene Expression Analysis
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Real-time PCR was performed with a PowerUp™ SYBR® Green Master Mix (Thermo Fisher Scientific). One microliter of cDNA (16 ng) was amplified with 500 nM each of the specific primer combinations for target genes, metallothionein-2 (mt2) and ribosomal protein L7 (rpl7), and 5 μL 2 × PowerUp™ SYBR® Green Master Mix in a total volume of 10 μL. PCR conditions were as follows: 40 cycles of 95°C for 3 sec and 60°C for 30 sec. Real-time PCR analysis of each sample was carried out according to a relative standard curve method using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Standard curves were obtained from serially diluted sample mixtures, and the expression levels of samples were measured using the standard curves. The rpl7 was chosen as an endogenous control, because its expression level is stable between fish and is unrelated to treatments (Lang et al., 2016) . Primers for the respective target genes (listed in Table 1) were designed to anneal at 60°C.
6
Quantifying Gene Expression using qPCR
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Quantitative PCR (qPCR) reactions were performed using PowerUp SYBR Green Master Mix (Applied Biosystems). Each qPCR reaction contained 10 ng of cDNA as template, 300 nM of each primer (Table 2
7
Quantitative PCR Assay Protocol
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Internal primers for use with SYBR dye-based qPCR assays were designed using Primer3 software for both pkd1 and myosin genes. All qPCR assays were performed on an Applied Biosystems QuantStudio 12. Concentrations for the plasmid preps were measured using the Qubit dsDNA BR assay kit (Invitrogen). A fresh tenfold serial dilution ranging over six logs (106 to 10 gene copy number (GCN)) of both the pTOPO-pkd1 and pTOPO-myosin plasmids were used to generate each standard curve (Table 1 ). A 10μL qPCR mixture was prepared using the PowerUp SYBR Green Master Mix (Invitrogen): 1X PowerUp SYBR Green Master Mix, 0.3μM forward and reverse primers, and 2μL template DNA or plasmid standards. The thermal cycling protocol was as follows:
95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min.
Following amplification, a melting curve analysis was used to confirm reaction specificity; a single and specific peak was generated for each primer pair. Both negative and no-template controls were performed in triplicate.
95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min.
Following amplification, a melting curve analysis was used to confirm reaction specificity; a single and specific peak was generated for each primer pair. Both negative and no-template controls were performed in triplicate.
8
Transcriptional Response to Nickel Stress
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Bacterial cultures of wild type and ΔnikR strains were grown to OD600 of 1.0–1.1 and split into 2 sub–cultures of 5 ml each that were treated either with 500 μM nickel (ni+) or with the same volume of sterile water (ni−) for 20 minutes. Samples were stopped by addition of 625 μl RNA stop solution (95% ethanol, 5% acid phenol) and RNAs were purified using 1 ml of Tri–reagent (Sigma-Aldrich) for each sample, following the manufacturer instructions. RNAs were treated with DNaseI prior to cDNA synthesis55 (link). Two μl of 1:10 diluted cDNAs were added to 5 μl of PowerUp™ SYBR® Green Master Mix (TermoFisher) and 400 nM of forward and reverse oligonucleotides in a 10 μl final volume. The qRT–PCR program was performed as previously described55 (link). Primer extension analysis was performed using 15 μg of total RNA and 0.1 pmol of radiolabeled probe. Northern blot assay was performed using 17 μg of total RNA and 1.25 pmol of radiolabeled oligo probe.
9
Investigating RpoE-Dependent EspPΔ5 Assembly
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AD202 transformed with pJH208 and either a reconstructed version of pLC45 (68 (link)) or pTrc99A were grown in M9 medium as described above. When cultures reached an OD550 of ∼0.2, they were treated with 10 μM IPTG for 25 min to induce rpoE expression and then 0.2% rhamnose for 5 min to induce EspPΔ5Y1298A, F1300A synthesis. EspPΔ5Y1298A, F1300A assembly was analyzed by pulse-chase labeling as described above, and samples were collected for qRT-PCR and Western blotting. For qRT-PCR, cells (1 OD550 equivalent) were collected, and RNA was prepared by using an RNeasy minikit (Qiagen). RNA preps were treated with DNase I (NEB) and used as the templates to synthesize cDNA using the SuperScript III first-strand synthesis system (Thermal Fisher). qPCR was performed using Power-Up SYBR Green Master Mix (Thermal Fisher) and Bio-Rad CFX-96 real-time PCR detection system to determine the expression of rpoE and the housekeeping gene rssA as a normalization reference. For Western blots, proteins were TCA precipitated.
10
Real-Time qPCR Validation of mRNA and miRNA Sequencing
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We choose the four mRNAs and four miRNAs to confirm the sequencing results. Real-time quantitative PCR was performed on a QuantStudio™ real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primers were designed by Oligo 6.0 and synthesized by the BGI (Beijing Genomics institution, Beijing, China). All of the primers used in the validation assays are listed in Supplementary Materials Table S1 . We used PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) to make a 10 μL PCR system, which consisted of 5 μL PowerUp™ SYBR™ Green Master Mix (2×), 0.3 μL upstream and downstream primers for each, 3 μL cDNA template, and 4.4 μL deionized distilled water. The reaction conditions were as follows: 2 min at 50.0 °C, 2 min at 95.0 °C, 40 cycles for 15 s at 95 °C, 15 s at 60 °C, and 1 min at 72 °C.
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