All samples and no-template controls for each assay were run in duplicate. Cycle quantification values (Cq) were excluded from analysis if they did not meet the quality control criteria in QuantStudio 7 Flex Software v.2.1 (Thermo Fisher) or if they were identified as an outlier. To obtain serum levels of the miRNAs of interest, the ΔCt approach (2−ΔCt algorithm) was used. The results are presented as the average of relative values ± SD, calculated as 2 − (Ct of miRNA of interest − Ct of normalizer). From the available approaches for normalization, exogenous reference cel-miR-39 was used as a normalizer for circulating miRNAs [39 (link),40 (link)].
Quantstudio 7 flex software
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 7 Flex software is a graphical user interface that enables users to control and monitor the QuantStudio 7 Flex Real-Time PCR System. It provides functions for experimental setup, data collection, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (3 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Quantstudio 7 flex real time qpcr, by Thermo Fisher Scientific (2 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Nanodrop onec, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Dnase digestion, by Qiagen (1 mentions)
Mircury lna rt kit, by Qiagen (1 mentions)
Mirna mimic, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Sybr green luna universal qpcr master mix, by New England Biolabs (1 mentions)
7 protocols using quantstudio 7 flex software
1
Quantifying Circulating miRNA Levels
2
Quantitative Gene Expression Analysis by TaqMan qRT-PCR
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Cells used for PCR validation were lysed in RLT Buffer with 1% 2-Mercaptoethanol (Qiagen). Total RNA was reverse transcribed using Superscript VILO Master Mix (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using TaqMan Universal PCR Master Mix with pre-validated TaqMan gene expression assays (Thermo Fisher). TaqMan assays were BST1 (Hs01070189_m1), TGM2 (Hs01096681_m1), MEST (Hs00853380_g1), CXCR4 (Hs00607978_s1), NES (Hs04187831_g1), TLR2 (Hs00610101_m1), HGF (Hs00300159_m1), SERPINF1 (Hs01106937_m1), CSF1 (Hs00174164_m1), CCL2 (Hs00234140_m1), CCL5 (Hs99999048_m1), MMP14 (Hs01037003_g1), TNFAIP6 (Hs00200180_m1), TIMP1 (Hs01092512_g1), SPP1 (Hs00959010_m1), C3 (Hs00163811_m1), C7 (Hs00940408_m1), CFI (Hs00989715_m1), C1R (Hs00354278_m1), C3AR1 (Hs00269693_s1), A2M (Hs00929971_m1) and endogenous control IPO8 (Hs00183533_m1). PCR was performed using QuantStudio 7 Flex real-time qPCR (Thermo Fisher). Relative changes in gene expression were determined by ΔΔCt method with QuantStudio 7 Flex software (Thermo Fisher).
3
Quantitative RT-PCR Analysis of Oligodendrocyte Genes
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Total RNA was isolated using the RNeasy Mini Kit (Qiagen). Cells were lysed in RLT Buffer with 1% 2-Mercaptoethanol (Millipore). On column DNase digestion was performed (Qiagen). Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen). mRNAs were quantified using a Nanodrop (One C, ThermoFisher Scientific). Quantitative real time PCR (qRT-PCR) was performed using TaqMan™ Fast Advanced Master Mix (ThermoFisher Scientific) with pre-validated TaqMan gene expression assays (ThermoFisher Scientific). TaqMan assays for human were olig2 (Hs00300164_s1), ng2 (Hs00361541_g1) and GalC (Hs01012300_m1). The expression of each gene was normalized to actin-beta (Hs01060665_g1) as a housekeeping gene. PCR was performed using QuantStudio 7 Flex real time qPCR (ThermoFisher Scientific). Relative changes in gene expression were determined by ΔΔCt method with QuantStudio 7 Flex software (ThermoFisher Scientific).
4
PRRSV Detection in Meat Samples
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Viral RNAs, extracted from meat juice, were subjected to qualitative RT-qPCR, using VetMAX™ PRRSV EU & NA 2.0 kit (Applied Biosystems, Foster City, CA, USA), targeting the PRRSV-1 (EU) and PRRSV-2 (NA) strains, following the manufacturer’s instructions. The samples were tested in duplicate.
RT-qPCR amplification was performed using a QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific) using the following thermal cycling conditions: a retro-transcription step at 50 °C for 5 min, an initial step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. The results were analyzed using the QuantStudio™ 7 Flex Software (Thermo Fisher Scientific). Positive and negative controls were introduced in each analytical session. In particular, negative controls for the extraction and amplification steps were included, and, in addition, an external positive control (EPC) and an internal positive control (IPC) provided by the abovementioned kit (Applied Biosystems) were used, following the manufacturer’s instructions. Positive and negative results were assigned to the analyzed samples as indicated by the kit datasheet: positive samples (Cq < 40) and negative samples (Cq > 40).
RT-qPCR amplification was performed using a QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific) using the following thermal cycling conditions: a retro-transcription step at 50 °C for 5 min, an initial step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. The results were analyzed using the QuantStudio™ 7 Flex Software (Thermo Fisher Scientific). Positive and negative controls were introduced in each analytical session. In particular, negative controls for the extraction and amplification steps were included, and, in addition, an external positive control (EPC) and an internal positive control (IPC) provided by the abovementioned kit (Applied Biosystems) were used, following the manufacturer’s instructions. Positive and negative results were assigned to the analyzed samples as indicated by the kit datasheet: positive samples (Cq < 40) and negative samples (Cq > 40).
5
qRT-PCR Analysis of Immune Gene Expression
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Cells were lysed in RLT buffer with 0.01% 2-mercaptoethanol (Qiagen, MD, USA). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse-transcribed using Superscript VILO Master Mix (Life Technologies). qRT-PCR was performed using TaqMan ® Universal PCR Master Mix with prevalidated TaqMan gene expression assays (Thermo Fisher). TaqMan assays for mouse cells were Ccl2 (Mm00441242_m1),Ccl8 (Mm01297183_m1),Nos2 (Mm00440502_m1),Arg1 (Mm00475988_m1) and Hprt (endogenous control) (Mm03024075_m1). Human TaqMan assays were CCL2 (Hs00234140_m1), CCL5 (Hs00982282_m1), CCL8 (Hs04187715_m1), CD206 (Hs00267207_m1), TGFB1 (Hs00998133_m1), TGFB2 (Hs00234244_m1), TGFB3 (Hs01086000_m1), IL6 (Hs00174131_m1), CX3CL1 (Hs00171086_m1) and endogenous control IPO8 (Hs00183533_m1). PCR was performed using QuantStudio™ 7 Flex real time qPCR (Thermo Fisher). Relative changes in gene expression were determined by the ΔΔCt method with QuantStudio 7 Flex software (Thermo Fisher).
6
Plasma miRNA Quantification by qPCR
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After RNA extraction from plasma and miRNA (2 μL/sample) conversion to cDNA (miRCURY LNA RT kit, Qiagen), quantitative real‐time PCR (qPCR, cDNA dilution 1:40) was performed (QuantStudio 7 Flex real‐time PCR system, Applied Biosystems) using miRCURY LNA miRNA PCR assay system following the manufacturer's instructions. A standard curve using specific miRNA mimics (miRNA mimics, Qiagen) and melting curve analysis were included in all reactions. The UniSp6 cycle threshold value (±0.2) was used as a quality control for inclusion in the data analysis. Patient information was protected by relabeling all samples and blinding the investigator. All samples were assayed in triplicate. QuantStudio™ 7 Flex software was used for data analysis (Applied Biosystems).
7
Liver RNA Isolation and qPCR Analysis
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RNA was isolated from livers homogenized in Buffer M using Trizol (Invitrogen, USA, 15239794), cDNA prepared using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA, 4368814) and qPCR performed using the SYBR Green Luna® Universal qPCR Master Mix (NEB, USA, M3003) on the QuantStudio™ 7 Flex
Real-Time PCR System (Applied Biosystems™, USA, 4485701). Primers were designed with the NCBI primer designing tool (primer-blast) and are listed in table 1.
Relative mRNA levels were analyzed as described by Schmittgen et al., (Schmittgen & Livak, 2008) (link) with modifications, as follows. The relative PCR-efficiency of the gene of interest (GOI) primer sets compared to the 18s rRNA (HKG) primer set was determined by computational standard curve analysis in QuantStudio™ 7 Flex Software, Applied Biosystems™, USA) and subsequent division of GOI/HKG primer efficiencies. The resulting value was used as a base to compute relative quantities from delta CT values.
Real-Time PCR System (Applied Biosystems™, USA, 4485701). Primers were designed with the NCBI primer designing tool (primer-blast) and are listed in table 1.
Relative mRNA levels were analyzed as described by Schmittgen et al., (Schmittgen & Livak, 2008) (link) with modifications, as follows. The relative PCR-efficiency of the gene of interest (GOI) primer sets compared to the 18s rRNA (HKG) primer set was determined by computational standard curve analysis in QuantStudio™ 7 Flex Software, Applied Biosystems™, USA) and subsequent division of GOI/HKG primer efficiencies. The resulting value was used as a base to compute relative quantities from delta CT values.
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