Total RNA was isolated using the NucleoSpin® RNA kit (Macherey-Nagel) and reverse transcribed with M-MLV reverse transcriptase (Thermo Fisher Scientific). Real-time PCR was performed using the PerfeCTa SYBR Green SuperMix low ROX (Quantabio) on a ViiA 7TM Real-Time PCR System (Thermo Fisher Scientific). Fold induction of Zfp597 was calculated relative to Rpl19 whereas Stat1 was compared to Actb by using the 2-ΔΔCt method. Standard curves of all primers were performed by testing serial dilutions of cDNA-experimental samples obtaining an average of 100 ± 5% efficiency. Correlation between target and housekeeping genes was assessed by standard curve comparisons (Zfp597-Rpl19 slope 0.0194 / Stat1-Actin slope 0.0188). Details about the primers used can be found as Supplementary Information.
Perfecta sybr green supermix low rox
Manufactured by Quantabio
Sourced in United States
PerfeCTa SYBR Green SuperMix Low ROX is a ready-to-use reaction mix for quantitative real-time PCR (qPCR) assays. It contains all the necessary components, including a chemically modified hot-start Taq DNA polymerase, SYBR Green I dye, and optimized buffer components.
Automatically generated - may contain errors
Lab products found in correlation
M mlv reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Nucleospin rna kit, by Macherey-Nagel (3 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Qscript cdna synthesis kit, by Quantabio (2 mentions)
Viia 7tm real time pcr system, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Qscript reverse transcription kit, by Quantabio (1 mentions)
Trizol, by MP Biomedicals (1 mentions)
Fastprep 24 5g homogenizer, by MP Biomedicals (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Qscript mirna cdna synthesis kit, by Quantabio (1 mentions)
Quantstudio 6 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Qscript microrna synthesis kit, by Quantabio (1 mentions)
Cfx96 thermocycler, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
12 protocols using perfecta sybr green supermix low rox
1
Gene Expression Analysis by RT-qPCR
2
RT-PCR Analysis of Colon Gene Expression
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Colon samples were collected and frozen at -80ºC. The day before the RNA extraction, 200 µl of RNAlater solution was added to the samples and stored at -20ºC. Total RNA from colon tissue was extracted using RiboZol and the Nucleospin RNA kit (Macherey–Nagel) according to the manufacturer`s protocol. M-MLV reverse transcriptase (ThermoFisher Scientific) was used to synthesize cDNA. RT-PCR was performed on 384 well plates in a QuantStudio 6 Flex Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with PerfeCTa SYBR Green SuperMix Low ROX (Quantabio, Beverly, MA, USA) and amplification was analyzed by QuantStudio RT PCR software v1.3. Primers for Ifng, Il1b, Lcn2, Mcj, Myd88,Timp3, Tlr4, Tlr5, Tlr9, Tnf, Tnfr1, Reg3b and Rpl19 genes were optimized (see sequences and annealing temperature in supplementary table S2 ). To normalize mRNA expression, the expression of 3 housekeeping genes was measured and Rpl19 was ranked as the best candidate. The mRNA relative quantification was calculated using the ΔΔCt method11 (link). PCR efficiency was always between 90 and 110%.
3
Quantitative Analysis of Viral and Inflammatory Markers in Tissue Samples
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Total RNA was extracted from tissues using Trizol (Ambion, 15596018). Tissues were homogenized into Trizol reagent using the FastPrep-24 5 G homogenizer (MP Biomedicals). Total RNA was isolated using the PureLink RNA mini-kit (Ambion, 12183025). Purified RNA was quantified, checked for quality assurance, and then reverse transcribed with the qScript Reverse Transcription kit (QuantaBio, 95047). For quantification of vRNA, a standard curve was generated using 10-fold serial dilutions of ZIKV RNA standard. qRT-PCR for ZIKV prM-E was performed with TaqMan Gene Expression Master Mix, ZIKV Primers, and probe as previously described.35 (link) The standard curve had an R value greater than 0.99. vRNA copies were interpolated from the standard curve using the average Ct value obtained from samples run in triplicate. qRT-PCR for GAPDH, IL-1β, TNF-α, IL-10, SOCS3, MMP2, MMP9, and COX2 were performed with PerfeCTa SYBR Green SuperMix Low Rox (Quantabio, #95056-500). Primers for GAPDH, IL-1β, TNF-α, IL-10, SOCS3, and COX2 were as previously described.36 (link) MMP2 primers were as follows; forward 5ʹ-AACGGTCGGGAATACAGCAG-3ʹ; reverse 5ʹ-GTAAACAAGGCTTCATGGGGG-3ʹ. MMP9 primers were as follows; forward 5ʹ-AACCTCCAACCTCACGGACA-3ʹ; reverse 5ʹ- AGGTTTGGAATCGACCCACG-3ʹ. Data was analyzed using the ∆∆Ct method to determine normalized relative expression.
4
Circulating miRNA Profiling for Metabolic Disorders
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A panel of 16 miRNAs was selected (Supplementary Table 1 ) due to published association with lipid and glucose metabolism and related disorders [23 (link)–25 (link), 30 (link), 32 (link)]. Circulating miRNA expression was analyzed after purification of RNA from serum using the miRNeasy Kit (Qiagen, Hilden, Germany). cDNA was synthesized with qScript miRNA cDNA Synthesis Kit (Quantabio, Beverly, MA, USA). miRNA quantification was completed by using PerfeCTa SYBRGreen SuperMix Low ROX (Quantabio) in real-time qRT-PCR. Supplementary to the commercially available kits, oligonucleotides for selected miRNAs (Supplementary Table 1 ) were purchased from Eurofins. Obtained CT values of miRs of interest were multiplied with −1 and normalized to the mean of CT of miRNAs 16, 24, 25, and 26 that served as internal controls [42 (link), 43 (link)]. Obtained −ΔCT values give the logarithmic relative expression.
5
Real-Time PCR for Gene Expression Analysis
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RNA was reverse transcribed using M-MLV reverse transcriptase (Thermo Fisher Scientific). Real-time PCR was then performed using the PerfeCTa SYBR Green SuperMix low ROX (Quantabio, Beverly, Massachusetts, USA) on a QuantStudio 6 Real-Time PCR System (Thermo Fisher Scientific). Fold induction of the genes was calculated relative to Rpl19 using the 2−ΔΔCt method. The primers used are listed in S4 Table .
6
Quantitative Gene Expression Analysis
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Total RNA was extracted using the TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) in accordance with the manufacturer’s instructions. cDNA was synthesized from the total RNA (1000 ng) by using the qScript cDNA synthesis kit (Quantabio, Beverly, MA, USA). miRNA cDNA was obtained from the purified total RNA (700 ng) using the qScript microRNA Synthesis Kit (Quantabio). Quantitative polymerase chain reaction (qPCR) for mRNA expression was performed with LightCycler 480 SYBR Green I Master (Roche Diagnostics, Indianapolis, IN, USA) and for miR-210 with PerfeCTa SYBR Green SuperMix, Low ROX (Quantabio), as previously described [19 (link),57 (link)]. Each PCR reaction was performed in duplicate. The PerfeCTa microRNA assay included Universal Primer, miR-210 Primer, and SNORD44 as positive control primers (Quantabio). The expression levels were normalized to beta-actin (ACTB; for mRNA) and ribosomal protein S18 (RPS18; for miRNA). The sequences of primers used for qPCR are listed in Table 3 . The threshold cycle (Ct) values of each sample were generated, and the relative expression was calculated as 2-ΔCt=2-(Ct target gene − Ct housekeeping gene) [58 (link)].
7
Quantitative RT-PCR Analysis of SALL1 Transcripts
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HEK 293FT cells transfected with 5 μg of CMV-SALL1-YPF, CMV-SALL1ΔSUMO-YFP or CMV-GFP-β-galactosidase plasmids, or HEK 293-TripZ-SALL1-2xHA_puro cells induced with different concentrations of doxycycline (dox), were used for reverse transcription-quantitative PCR (RT-qPCR) analysis. Forty-eight hours after transfection, or 72 h after induction, total RNA was obtained by using EZNA Total RNA Kit (Omega) and quantified using a NanoDrop spectrophotometry. cDNAs were prepared using the SuperScript III First-Strand Synthesis System (Invitrogen) using 1 mg of total RNA in 20 μl volume per reaction. qPCR was done using PerfeCTa SYBR Green SuperMix Low Rox (Quantabio). Reactions were performed in 20 μl, adding 5 μl of cDNA and 0.5 μl of each primer (10 μM), in a CFX96 thermocycler (BioRad) using the following protocol: 95°C for 5 min and 40 cycles of 95°C for 15 s, 56 or 62°C for 30 s and 72°C 20 s. Melting curve analysis was performed for each pair of primers between 65 and 95°C, with 0.5°C temperature increments every 5 s. Relative gene expression data were analyzed using the ΔΔCt method (Livak and Schmittgen, 2001 (link)). Reactions were carried out in duplicate and results were derived from at least three independent experiments, normalized to GAPDH and presented as relative expression levels. Primer sequences are listed in Table 2 .
8
Quantitative PCR Protocol for Measuring mRNA Levels
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RNA was isolated from muscle homogenates and reverse transcribed to produce cDNA24 (link). QPCR experiments were designed using established guidelines for experimental design, data normalization and data analysis to maximize the rigor of quantifying the relative levels of mRNA13 (link),66 (link),67 (link). The expression for each gene in control samples was set to 1 and the other expression values were then scaled to that value. PCR primers are listed in Supplementary Table 1 .
Cultured cells were washed twice with ice-cold DPBS and collected in Trizol (Invitrogen). RNA was extracted and isolated with chloroform extraction and isopropyl alcohol precipitation, followed by clean-up with an RNA Clean and Concentrator Kit (Zymo Research). Total RNA was quantified, reverse transcribed, and used for QPCR13 (link).
RNA was isolated from FACS sorted cells by first sorting cells directly into Buffer RLT RNA lysis buffer (Qiagen). RNA was isolated using a Qiagen RNeasy Micro Kit according to the manufacturer’s protocol. RNA yield was quantified using a BioDrop μLite. RNA (50 ng/reaction) was reverse transcribed using a qScript XLT cDNA Supermix (QuantaBio). QPCR experiments were performed on a QuantStudio 5 Real-Time PCR System (Thermo Fisher) with PerfeCTa SYBR Green Supermix, Low Rox (QuantaBio)13 (link).
Cultured cells were washed twice with ice-cold DPBS and collected in Trizol (Invitrogen). RNA was extracted and isolated with chloroform extraction and isopropyl alcohol precipitation, followed by clean-up with an RNA Clean and Concentrator Kit (Zymo Research). Total RNA was quantified, reverse transcribed, and used for QPCR13 (link).
RNA was isolated from FACS sorted cells by first sorting cells directly into Buffer RLT RNA lysis buffer (Qiagen). RNA was isolated using a Qiagen RNeasy Micro Kit according to the manufacturer’s protocol. RNA yield was quantified using a BioDrop μLite. RNA (50 ng/reaction) was reverse transcribed using a qScript XLT cDNA Supermix (QuantaBio). QPCR experiments were performed on a QuantStudio 5 Real-Time PCR System (Thermo Fisher) with PerfeCTa SYBR Green Supermix, Low Rox (QuantaBio)13 (link).
9
Quantitative Analysis of Osteogenesis
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Cultured cells were incubated in 6‐well plates to ensure sufficient RNA was extracted. Total RNA extraction was performed using RNeasy kit (Qiagen, Hilden, Germany) and analysed using NP80 Nanophotometer (Implen, Munich, Germany). Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Walham, MA, USA) was used before qPCR was performed using PerfeCTa SYBR® Green SuperMix Low ROX (Quantabio, Beverly, MA, USA). Data were analysed using QuantStudio 7 Flex Real‐Time PCR System (Applied Biosystems, Waltham, MA, USA) and relative expression was calculated according to the 2ΔΔCt method using GAPDH as a reference gene. Gene expression for osteogenic marker Runt‐related transcription factor 2 (Runx2) was analysed.
10
Colon Tissue RNA Extraction and qPCR Analysis
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Colon samples were collected and frozen at −80 °C. The previous day of the RNA extraction, 1 ml of RNAlater solution was added to the samples and stored at −20 °C. Total RNA from colon tissue was extracted using TRIzol (Invitrogen) and Nucleospin RNA kit (Macherey-Nagel) according to the manufacturer’s protocol. M-MLV reverse transcriptase (ThermoFisher Scientific) was used to synthesize cDNA. Real-time PCR (qPCR) was performed on 384 well plates by QuantStudio 6 Flex Real-Time PCR system (Thermo Fisher Scientific) with PerfeCTa SYBR Green SuperMix Low ROX (Quantabio) and amplification was analyzed by QuantStudio Real-Time PCR software v1.3. Primers for Il1b, Lcn2, Myd88, Reg3b, Rpl19, Tnf and Tnfr1 genes were optimized (see sequences and annealing temperature in Supplementary Table 1 ). To normalize mRNA expression, the expression of 3 housekeeping genes was measured and Rpl19 was ranked as the best candidate. The mRNA relative quantification was calculated using the ΔΔCt method38 (link), PCR efficiency was always between 90 and 110%.
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