Quantification of mRNA expression of target genes was performed as described previously [16 (link)]. Total RNAs of skeletal muscle and C2C12 cells were isolated by using Trizol (Invitrogen), followed by purification with NucleoSpin RNA Clean-up (MACHEREY-NAGEL). Complementary DNA was synthesized from 0.5–1 μg total RNA using PrimeScript RT Reagent kit with gDNA Eraser (TaKaRa). PCR reactions were prepared by using KAPA SYBR FAST Master Mix Universal (KAPA BIOSYSTEMS) followed by the real-time PCR using Thermal Cycler Dice (TaKaRa). Nucleotide sequence of each primer is shown in Table 1 . mRNA levels for target genes relative to 18S rRNA or actin was shown for all the experiments.
Kapa sybr fast universal master mix
Manufactured by Roche
Sourced in United Kingdom, United States
KAPA SYBR Fast Universal Master Mix is a pre-formulated, ready-to-use qPCR solution designed for fast, sensitive, and reproducible real-time PCR amplification. The master mix contains KAPA SYBR FAST DNA polymerase, proprietary buffer, and PCR enhancers for efficient amplification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Thermal cycler dice, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna clean up, by Macherey-Nagel (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Escherichia coli top10 cells, by Thermo Fisher Scientific (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Roche (1 mentions)
Nanodrop spectrometer, by Thermo Fisher Scientific (1 mentions)
Cfx384 instrument, by Bio-Rad (1 mentions)
Rt2 ht first strand kit, by Qiagen (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Rt2 qpcr primer pairs, by Qiagen (1 mentions)
Peqgold total rna kit, by Avantor (1 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Roche (1 mentions)
Qbaseplus software, by CellCarta (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
10 protocols using kapa sybr fast universal master mix
1
Quantitative mRNA Expression Analysis
2
Validation of Editing Site Identification
3
Cholesterol Regulation of Gene Expression
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Total RNA was extracted from cholesterol treated and untreated BRIN-BD11 cells using TRIzol reagent (Life Technologies) and 1 μg of total RNA was used to synthesize cDNA using Superscript III First-Strand cDNA synthesis kit. Quantitative real-time PCR was performed in the Step One -Plus Real -Time PCR system using kapa sybr fast universal master mix (Kapa Biosystems). The relative abundance of the mRNAs was measured using 2−ΔΔCT 48 with GAPDH mRNA as invariant reference.
4
Retinal RNA Isolation and Quantification
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The retina was isolated and flash frozen in liquid nitrogen. The RNeasy mini kit (74106; Qiagen) was used to isolate RNA from the whole retina. A Nanodrop spectrometer (ThermoScientific) was used to determine the RNA concentration of the samples. Five hundred nanograms of RNA was synthesized into cDNA by Superscript III (18080-044; ThermoScientific-Invitrogen). Absolute quantification RT-PCR for whole retina was performed with KAPA SYBR Fast Universal Master Mix (KK4600; KAPA Biosystems) on a Step One Plus real-time PCR system (ThermoScientific-Applied Biosystems).
5
Gene Expression Profiling in Infected Cells
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In infected LoVo or JEG-3 cells, RNA was extracted at, respectively, 18 or 20 h p.i. with an RNeasy minikit (Qiagen) as recommended by the manufacturer, using 1 column per well of a 24-well plate and biological duplicates for each condition. Genomic DNA was removed by treatment with a Turbo DNA-free kit (Ambion). cDNAs were generated from 1 to 2 µg total RNA using the RT2 HT first-strand kit (Qiagen/SABiosciences).
qPCR was performed on a CFX384 instrument (Bio-Rad) using Kapa SYBR Fast universal master mix (Kapa Biosystems) as specified by the supplier. Each reaction was performed in triplicate. RT-qPCR primers for YWHAZ, IFI44L, IFITM1, and IL6 were predesigned, validated RT2 qPCR primer pairs from Qiagen/SABiosciences. Other primer pairs were selected from the literature, from primer databases (RTPrimerDB,http://medgen.ugent.be/rtprimerdb/ , or qPrimerDepot, http://primerdepot.nci.nih.gov/ ) and validated for efficiency. Data were analyzed by the ∆∆CT method. Target gene expression data were normalized to the relative expression of the YWHAZ reference gene. The displayed results are representative of three independent experiments in LoVo cells and two experiments in JEG-3 cells.
qPCR was performed on a CFX384 instrument (Bio-Rad) using Kapa SYBR Fast universal master mix (Kapa Biosystems) as specified by the supplier. Each reaction was performed in triplicate. RT-qPCR primers for YWHAZ, IFI44L, IFITM1, and IL6 were predesigned, validated RT2 qPCR primer pairs from Qiagen/SABiosciences. Other primer pairs were selected from the literature, from primer databases (RTPrimerDB,
6
Quantitative Analysis of MSC and Neuronal Markers
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Quantitative real-time PCR (qRT-PCR) was used to quantify mRNA expression of MSC and neuronal markers as well as different connexin isoforms. Undifferentiated MSCs were used as control. RNA was isolated using the PeqGOLD Total RNA kit (Peqlab). Thereafter, RNA was reverse transcribed into cDNA using the Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, United States). 12.5 ng of cDNA were used as template for the qRT-PCR which was performed with the KAPA SYBR FAST Universal mastermix (Kapa Biosystems) in a volume of 10 μL. All primer data are given in Supplementary Table 3 . Correct amplification was confirmed by sequencing. For each sample and primer pair three technical replicates were analyzed. Each qRT-PCR was run at least three times with cDNA of independent differentiations. The mRNA level of the gene of interest relative to the housekeeping gene RPS29 was calculated by 2–ΔCt method.
7
Detecting Human DNA in Sand Flies
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Female sand flies identified as P. argentipes by PCR-RFLP were analysed for the presence of human DNA using a qPCR protocol with primers targeting the cytochrome b (cytb) gene as described previously [25 (link)]. Quantitative detection of human DNA was performed on an Applied Biosystems 7500 Fast Real-Time PCR System according to the KAPA SYBR FAST Universal Master Mix recommended protocol. For standard curves, human DNA obtained from purchased donor blood (Cambridge Bioscience) was serially diluted to provide a range of 1–0.0001ng/μl. DNA free water was used as No Template Control (NTC) in each assay. A total of 5μl of DNA was used from each female P. argentipes. Samples were considered positive if Ct values were lower than the lower limit of detection of the assay (Ct< 30). All samples and controls were run in duplicate.
8
Quantitative mRNA Expression Analysis
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Total RNA was extracted from BRIN-BD11 cells, cultured human adipocytes, and mouse adipose tissues using TriZol reagent (Life Technologies), and 1 µg of total RNA was used to synthesize cDNA using Superscript III First-Strand cDNA synthesis kit. Specific mRNA was amplified and quantified by quantitative real-time PCR using Quant Studio 5 (A and B Biosystems). Kapa sybr fast universal master mix (Kapa Biosystems) reagent was used to assess the relative abundance of the mRNAs measured using the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)). Data were normalized using 18s rRNA as an invariant reference in adipocytes and GAPDH in cultured pancreatic beta cells. Primers used for amplification are listed in Supplementary file 3 Table 3.
9
Quantitative PCR Analysis of mTOR Pathway
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Relative expression of mTOR, DEPTOR, rictor and raptor (Table I ) were assessed by quantitative PCR (Q-PCR) on an xxpress® (BJS Biotechnologies, Middlesex, UK) thermal cycler using Kapa SYBR Fast Universal Mastermix (Kapa Biosystems, MA, USA). According to MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines (11 (link)), an assessment of the most stably expressed reference genes specific to the samples used must be carried out prior to any qPCR experiment. In light of this, a selection of 8 ovarian clinical samples were assessed using the geNorm human 12 gene kit (Primer Design, Southampton, UK) according to the manufacturer's instructions. Reference gene expression stability was analysed using qbaseplus software (Biogazelle, Zwijnaarde, Belgium). Primers for mTOR, Deptor, Rictor and Raptor were used as previously described (10 (link)). qPCR data were analysed using the ΔCq method whereby the Cq of the endogenous control was subtracted from the Cq of the gene of interest and an RQ (relative quantity) value was calculated by finding 2−ΔCq (11 (link),12 (link)). Where more than one reference gene was used, the RQ values were averaged. A Student's t-test was used to calculate statistical significance.
10
Retinal Vascular Gene Expression Analysis
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RNA was isolated from the retinal vasculature and the GCL and collected by LCM. RNA isolation was performed with the RNeasy micro kit (74004; Qiagen, Valencia, CA, USA). Samples underwent cDNA synthesis by Superscript III (18080-044; Thermo Scientific-Invitrogen). RT-PCR was performed with KAPA SYBR Fast Universal Master Mix (KK4600; KAPA Biosystems, Wilmington, MA, USA) on a Step One Plus real-time PCR system (ThermoScientific-Applied Biosystems). Primers for Cd55 and Cd59 were created by the online Primer Quest design tool and bought from Integrated DNA Technologies (Coralville, IA, USA). Triplicate wells for each sample were run for RT-PCR, and the average threshold cycle (CT) value was used for analysis. All CT values were normalized to the internal control of β-actin. Final analysis was performed by the ∆∆CT method.
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