gDNA was extracted using the GenEluteTM Mammalian Genomic DNA miniprep Kit (Sigma, Overijse, Belgium) according to the manufacturer’s protocol. A total of 25 ng/µL gDNA was used for qPCR to determine integrated copies based on the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). As a control, a single integrated copy control (1ICC) was made generated by transducing HEK293T cells in a limiting dilution series with an LV expressing pEF1a-CTNSWT-eGFP-IRES-PuroR. Cells were monitored by flow cytometry analysis, and the condition where the Mean Fluorescence Intensity (MFI) did not drop when the vector was diluted, but the % eGFP positive cells did, was considered as the 1ICC condition. Following puromycin selection, the highest dilution that survived the selection was chosen. RT-qPCR was performed using 25 ng/µL gDNA, dsDNA-intercalating agent LightCycler® 480 SYBR Green I (Roche Life Science, Brussels, Belgium), and 10 µM primers (see Table S1 ). RT-qPCR was performed on the CFX Opus 96 Real-Time PCR instrument (Bio-Rad, Temse, Belgium) and data were retrieved and analysed using the CFX maestro 2.2 software. Amplification was performed for 50 cycles of 10 s at 95 °C and 30 s at 60 °C. The fold change was calculated as fold change = 2−∆∆Ct.
Cfx opus 96 real time pcr instrument
Manufactured by Bio-Rad
Sourced in Belgium, United States
The CFX Opus 96 Real-Time PCR Instrument is a thermal cycler designed for real-time PCR analysis. It features a 96-well format and can perform quantitative and qualitative gene expression analysis, genotyping, and other real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 sybr green 1, by Roche (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Kapa sybr fast qpcr master mix, by Bio-Rad (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Genelute mammalian genomic dna miniprep kit, by Merck Group (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Aurum total rna mini kit, by Bio-Rad (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Quantitect primer assay, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Gdna wiper mix, by Vazyme (1 mentions)
Hiscript 2 q rt supermix, by Vazyme (1 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (1 mentions)
Rna easy isolation reagent, by Vazyme (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
18 protocols using cfx opus 96 real time pcr instrument
1
Quantifying Viral Vector Integration
2
Quantitative RT-PCR in P. aeruginosa
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The QuantiTect Reverse Transcription Kit (Qiagen) was used for cDNA generation, following manufacturer instructions. qRT-PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA) on a CFX Opus 96 Real-Time PCR Instrument (Bio-Rad). Data were analyzed using the ΔΔCT method using rpsL, a P. aeruginosa ribosomal protein gene, as a control housekeeping gene. Primer sequences are listed in Table S5.
3
Quantification of CTNS mRNA Expression
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Total mRNA was extracted using the AurumTM Total RNA Mini Kit (Bio-rad, Temse, Belgium) following the manufacturers’ instructions. cDNA was synthesized from the extracted mRNA samples using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Merelbeke, Belgium). RT-qPCR was performed using 5 ng/µL cDNA, dsDNA-intercalating agent LightCycler® 480 SYBR Green I (Roche Life Science, Brussels, Belgium), and 10 µM primers. Primers were designed to land in exonic sequences, spanning exon 10 and 11, allowing for the assessment of endogenous mRNA- and LV-expressed CTNS mRNA (Table S1 ). RT-qPCR was performed on the CFX Opus 96 Real-Time PCR instrument (Bio-Rad, Temse, Belgium) and data were retrieved and analysed using the CFX maestro 2.2 software. Amplification was performed for 50 cycles of 10 s at 95 °C and 30 s at 60 °C. The fold change was calculated as fold change = 2−∆∆Ct.
4
Quantitative RT-PCR gene expression analysis
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RNA was isolated with RNeasy Mini Kits (Qiagen). Complementary DNA was synthesized using iScript cDNA synthesis Kit (Bio-Rad). Real-time qPCR was performed with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a CFX Opus 96 real-time PCR instrument (Bio-Rad), normalized to GAPDH. Primers were from QuantiTect Primer Assays (Qiagen).
5
Reverse Transcription and qPCR of TE-Containing Transcripts
6
Bacterial RNA Extraction and qPCR Quantification
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Exponentially growing bacterial cultures (optical density at a wavelength of 600 nm (OD600) 0.4–0.6) were pelleted. After the supernatant was removed, the cell pellet was resuspended in RNA-easy Isolation Reagent (Vazyme). The total RNA was precipitated by the addition of isopropanol and collected by centrifugation. The supernatant was discarded, and the RNA pellet was washed with 75% ethanol. After the pellet was air-dried, the mRNA was dissolved in RNase-free H2O. Any contaminating genomic DNA was digested with gDNA wiper Mix (Vazyme). The purified mRNA was reverse transcribed to cDNA with HiScript II qRT SuperMix (Vazyme). The cDNA levels of the target genes were then quantified with quantitative real-time PCR (qPCR) on CFX Opus 96 Real-Time PCR Instrument (Bio-Rad) using AceQ Universal SYBR qPCR Master Mix (Vazyme). All qPCR primers were determined to be >95% efficient, and the cDNA molecular masses were experimentally confirmed to be within the linear dynamic range of the assay. The signals were normalized to those of the housekeeping 16S rRNA transcript and quantified with the ΔΔCt method. The error bars are the 95% confidence intervals of three technical replicates.
7
RNA Extraction and qRT-PCR Analysis
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Total RNA from prepared samples was extracted using miRNeasy kit (Qiagen). Extracted RNA was then added to cDNA synthesis mix (SuperScriptTM VILOTM cDNA Synthesis Kit, Invitrogen) composed of 1× SuperScriptTM Enzyme Mix and 1× VILO™ Reaction Mix. cDNA was prepared with the following incubation protocol: 25 °C, 10 min; 42 °C, 60 min; 80 °C, 5 min for enzyme inactivation. As-prepared cDNA was added to qRT-PCR reaction mix composed of 1× TaqManTM Fast Advanced Master Mix (ThermoFisher Scientific) and 1× gene-specific TaqManTM Gene Expression Assay Mix (ThermoFisher Scientific). qRT-PCR was conducted on CFX Opus 96 real-time PCR instrument (Bio-Rad) with the following steps: 95 °C, 1 min and then subsequent thermal cycling schedule (55 cycles): 95 °C, 3 sec; 60 °C, 30 sec; fluorescence measurement. Setting the fluorescent threshold value to 400, quantification cycle (Cq) values were determined by the system software.
8
Quantifying Gene Expression by RT-qPCR
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Total RNA extraction was performed using the TRI Reagent (Merck, product code T9424) following the manufacturer’s instruction. For first-strand synthesis of cDNA, 1μg of RNA was used in a 20-μl reaction mixture by using a SensiFast cDNA synthesis kit (Bioline). For real-time PCR analysis, 0.4μL of cDNA sample was amplified by using the SensiFast Syber (Bioline) in an CFX Opus 96 Real-Time PCR Instrument (Bio-Rad Laboratories GmbH, Segrate, Italy). The reaction conditions were 95 °C for 5 min followed by 45 cycles of 15 sec at 95 °C and 1 min at 60 °C. The sequence of the oligos used are listed below in 5’-3’ direction:
9
Quantitative PCR Assay for Detection
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Each 15 µl qPCR reaction mixture contained 1 × TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific Inc.), 0.1 mg/ml bovine serum albumin (BSA) (Sigma–Aldrich), 250 nM of each primer, 100 nM of each TaqMan probe, 1 × TaqMan™ Exogenous Internal Positive Control (VIC-TAMRA labelled) Reagents (Thermo Fisher Scientific Inc.) and 2 µl template DNA. A negative control was included in each run, which consisted of nuclease-free water (W4502; Sigma–Aldrich). The qPCR program consisted of 2 min at 50 °C, 3 min at 95 °C, followed by 45 cycles of 95 °C for 3 s and 60 °C for 30 s. Amplification was performed in a CFX Opus 96 Real-Time PCR Instrument (Bio-Rad Laboratories Inc., Hercules, CA, USA) and analysed by the CFX Maestro Software version 2.0 (Bio-Rad Laboratories Inc.) with default settings. Samples for which a copy number equal to or above the limit of detection (LOD) was calculated from the qPCR assays were considered as positive.
10
RT-qPCR Protocol for Sensitive Quantification
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The reverse-transcription (RT)
reaction was performed in a 20 μL solution containing 1×
AMV buffer (New England Biolabs), 0.5 mM dNTPs (10 mM, New England
Biolabs), 10 U AMV reverse transcriptase (New England Biolabs), and
15 μL of pooled samples at 42 °C for 60 min and terminated
at 80 °C for 5 min. Then, RT products in the supernatant were
kept after magnetic separation. Finally, each PCR amplification reaction
was performed in a 25 μL solution consisting of 2.5 μL
of RT products, 800 nM universal forward primer, 40 nM of each reverse
primer, 10 μL of Premix Ex Taq (Takara Bio), 200 nM fluorophore
probe, and 200 nM quencher probe. PCR and melting curve analysis were
performed in a CFX Opus 96 real-time PCR instrument (Bio-Rad) as follows:
95 °C for 30 s and 50 cycles of 95 °C for 5 s, 60 °C
for 20 s, 95 °C for 1 min, 40 °C for 1 min, followed by
a temperature increase from 40 to 80 °C (0.5 °C/step). Fluorescence
intensity was measured in the FAM channel at each step of the continuous
temperature increase. The size of target amplicons was analyzed by
a 5200 Fragment Analyzer System (Agilent) and the Agilent DNF-905
dsDNA kit (1-500 bp).
reaction was performed in a 20 μL solution containing 1×
AMV buffer (New England Biolabs), 0.5 mM dNTPs (10 mM, New England
Biolabs), 10 U AMV reverse transcriptase (New England Biolabs), and
15 μL of pooled samples at 42 °C for 60 min and terminated
at 80 °C for 5 min. Then, RT products in the supernatant were
kept after magnetic separation. Finally, each PCR amplification reaction
was performed in a 25 μL solution consisting of 2.5 μL
of RT products, 800 nM universal forward primer, 40 nM of each reverse
primer, 10 μL of Premix Ex Taq (Takara Bio), 200 nM fluorophore
probe, and 200 nM quencher probe. PCR and melting curve analysis were
performed in a CFX Opus 96 real-time PCR instrument (Bio-Rad) as follows:
95 °C for 30 s and 50 cycles of 95 °C for 5 s, 60 °C
for 20 s, 95 °C for 1 min, 40 °C for 1 min, followed by
a temperature increase from 40 to 80 °C (0.5 °C/step). Fluorescence
intensity was measured in the FAM channel at each step of the continuous
temperature increase. The size of target amplicons was analyzed by
a 5200 Fragment Analyzer System (Agilent) and the Agilent DNF-905
dsDNA kit (1-500 bp).
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