Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, QIAGEN N.V. Corporate, Germany) and reverse-transcribed via the reverse transcriptase (Invitrogen, Invitrogen Corporation, United States) according to the manufacturer’s instructions. A total of 50 ng cDNA was used for quantitative PCR in each reaction with SYBR Green PCR Master Mix (Takara, TaKaRa Biotechnology (Dalian) Co.,Ltd., Japan), and then detected by using ABI real-time PCR detection system (Quantstudio 7, Quantstudio 7, Applied Biosystems, United States). Relative amounts of SCPL41 transcripts were calculated by the comparative cycle threshold method normalized to ACTIN2 expression from the same sample. Values were determined from three replicates.
Quantstudio 7
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, United Kingdom
The QuantStudio 7 is a real-time PCR system designed for quantitative gene expression analysis, genotyping, and copy number variation studies. It features a 96-well block format, a compact footprint, and supports a wide range of chemistries and sample volumes.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (23 mentions)
Trizol, by Thermo Fisher Scientific (20 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (12 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (11 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (8 mentions)
Rneasy kit, by Qiagen (7 mentions)
Rneasy plus mini kit, by Qiagen (7 mentions)
Iscript cdna synthesis kit, by Bio-Rad (6 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (6 mentions)
Quantitect reverse transcription kit, by Qiagen (5 mentions)
Rneasy micro kit, by Qiagen (5 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (5 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (5 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (4 mentions)
Sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Sybr select master mix, by Thermo Fisher Scientific (4 mentions)
Protoscript 2 reverse transcriptase, by New England Biolabs (4 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
209 protocols using quantstudio 7
1
Quantitative Analysis of SCPL41 Expression
2
SARS-CoV-2 Detection and Quantification in Autopsy
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qRT-PCR for detection and quantification of SARS-CoV-2 in autopsy samples were performed as described (Corman et al., 2020 (link)). Briefly, primers, probes and 5 μL of RNA were added to 10 μL of SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase mastermix (ThermoFisher). PCR was performed on a Quantstudio 7 instrument (ThermoFisher) with the following cycling conditions: 55 °C for 15 min, 95 °C for 3 min; 45 cycles consisting of 95 °C for 15 sec and 58 °C for 30 sec. Amplification data was downloaded and processed using the qpcR package of the R project (https://www.r-project.org/ ). Amplification efficiency plots were visually inspected and Cp2D (cycle peak of second derivative) values were calculated for samples with valid amplification curves. Plots were generated with R using the reshape, tidyverse and ggplot packages. qRT-PCR of virus cultures employed primer sets recommended by the CDC detecting three different regions of the viral nucleocapsid and human RNAseP or GAPDH as control (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html ). PCR was performed with the SYBR Green PCR Mastermix (Applied Biosystems) on a Quantstudio 7 instrument (ThermoFisher) with the following cycling conditions: 25 °C for 2 min, 50 °C for 15 min, 95 °C for 10 min, 45 cycles consisting of 95 °C for 3 sec and 55 °C for 30 sec.
3
Quantitative microRNA Expression Profiling
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RTqPCR was performed for miRs-122, miR-200a, let-7e and cel-miR-39 in 96 well plates on the Life Technologies Quantstudio7 machine according to manufacturer’s instructions. Briefly; one PCR reaction contained 0.5 µl of Taqman® primer/probe mix (Life Technologies), 10 µl of 2x Gene Expression Master-mix (Life Technologies), 6.5 µl of DNase/RNase free H2O and 3ul of diluted pre-amplification product.
For miR-200a and let-7e if cel-miR-39 and miR-122 was detected < 30 cycles in a sample but miR-200a or let-7e was not detected the sample was designated as having a Ct of 35 (the lower limit of detection) to signify extremely low expression or no expression of that particular miRNA. If cel-miR-39 and/or miR-122 was detected over 30 cycles and either miR-200a or let-7e wasn’t detected the RNA or extraction was considered unreliable and this sample was not used in further analyses.
For miR-200a and let-7e if cel-miR-39 and miR-122 was detected < 30 cycles in a sample but miR-200a or let-7e was not detected the sample was designated as having a Ct of 35 (the lower limit of detection) to signify extremely low expression or no expression of that particular miRNA. If cel-miR-39 and/or miR-122 was detected over 30 cycles and either miR-200a or let-7e wasn’t detected the RNA or extraction was considered unreliable and this sample was not used in further analyses.
4
Comprehensive RNA Expression Analysis
5
Identification of Mycobacterium bovis by RT-PCR
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Representative colonies were identified as M. bovis by their colony characteristics and by real time polymerase chain reaction (RT-PCR).24 (link)
PCR reaction was prepared using the following reagents: 0.375 nM of each primer (Mbovis.88.F: 5′-CGC CTT CCT AAC CAG AAT TG-3′ and Mbovis.88.R: 5′-GGA GAG CGC CGT TGT AGG-3′), 10 μL of Fast EvaGreen qPCR Master Mix (Biotium, USA) in a 20 μL reaction. Thermocyler (QuantStudio7, Life Technologies, USA) was programmed as follows: 95 °C for 5 min, followed by 35 cycles at 95 °C for 15 s, 63 °C for 20 s, and 72 °C for 30 s, with the reading cycle length. The curve denaturation was performed at 72–99 °C, with intervals of fluorescence at every 1% rise in temperature.
On egg media the typical M. bovis colony was small, rounded, pale yellow to buff with irregular edges and granular surface. On agar medium they were white, thin, rough and flat with a central mound.11 (link)
PCR reaction was prepared using the following reagents: 0.375 nM of each primer (Mbovis.88.F: 5′-CGC CTT CCT AAC CAG AAT TG-3′ and Mbovis.88.R: 5′-GGA GAG CGC CGT TGT AGG-3′), 10 μL of Fast EvaGreen qPCR Master Mix (Biotium, USA) in a 20 μL reaction. Thermocyler (QuantStudio7, Life Technologies, USA) was programmed as follows: 95 °C for 5 min, followed by 35 cycles at 95 °C for 15 s, 63 °C for 20 s, and 72 °C for 30 s, with the reading cycle length. The curve denaturation was performed at 72–99 °C, with intervals of fluorescence at every 1% rise in temperature.
On egg media the typical M. bovis colony was small, rounded, pale yellow to buff with irregular edges and granular surface. On agar medium they were white, thin, rough and flat with a central mound.11 (link)
6
Quantifying Mesenchymal and Neural Stem Cell Markers
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We examined the gene expression of a number of markers commonly used to define MSCs and neural stem cells (NSCs). These included markers for each of the three mesenchymal lineages (bone, cartilage and fat) as well as markers specific to NSCs and early and late neural development. For the genes of interest, mRNA levels were quantified using Q-PCR (Life Technologies QuantStudio 7). Briefly, 120ng cDNA was amplified with 200μM primers (forward and reverse) with SYBR-Green PCR Master Mix (Promega, Australia) in a 10μL reaction. Cycling conditions were as follows: 50°C for 2 min, 95°C for 3 min then 50 cycles of 95°C for 3s, 60°C for 30s. Gene expression was normalised against expression of 18S and calculated using 2-ΔΔCt. Specific primer sequences (IDT, USA) for the genes investigated can be found in S1 Table . Mean gene expression (2-ΔΔCt) was calculated between three independent experiments and graphed with SEM. All experiments were performed in quadruplicate.
7
Gene Expression Analysis by qRT-PCR
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cDNA was synthesized from the total RNA with the RT2 (link) First Strand Kit (Qiagen), Superscript VILO IV (Thermo Fisher), or iSCRIPT cDNA kit (BioRad), always with an additional DNase I treatment step. qRT-PCR assays were performed in technical duplicates in 96- or 384-well plates on QuantStudio 7 (Life Technologies) or Bio-Rad CFX 96 instrument, with RT² SYBR Green (Qiagen), POWER SYBR (Thermo Fisher), iTaq SYBR (BioRad) or TaqMan (Thermo Fisher) expression assays (Table S4 ). The expression of target genes was normalized by geometric means of endogenous controls (GAPDH, HPRT1, TBP or ACTB, as indicated in Table S2 ), and presented as dCt values relative to endogenous controls (log2 scale).
8
RNA-seq and qPCR Analysis of EDL Muscle
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At 6 and 12 weeks of age three control and three model mice were sacrificed, and EDL muscles were dissected, snap frozen in liquid nitrogen-cooled isopentane, and stored at −80°C. Total RNA was extracted using TRIzolTM Reagent (ThermoFisher Scientific) according to the manufacturer’s instructions. Contaminating genomic DNA was removed using the TURBO DNA-freeTM kit (ThermoFisher Scientific). Quality and concentration was assessed using a NanoDropTM (ThermoFisher Scientific) and Agilent Tapestation. RNA-seq was carried out by Novogene. See Supplementary material for analysis methodology. cDNA was generated from 2 µg RNA using a High-Capacity RNA-to-cDNA Kit (Life Technologies Ltd). qPCR was carried out on a QuantStudio7 (Life Technologies Ltd) using TaqMan Gene Expression Master Mix and Taqman Gene Expression Assay kits (Life Technologies Ltd). A list of the genes and probe IDs is given in Supplementary Table 1 . The internal gene control was Hprt1. Analysis methods are described in Supplementary material .
9
Quantitative Real-Time PCR for Gene Expression
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Total RNAs from mouse tissues were extracted using TRNzol according to the manufacturer's instructions. The first-strand DNA synthesis using total RNAs from mouse tissues as the templates was performed with 4 × EZscript Reverse Transcription Mix II. The standard material for calibration curves of coding sequence of mouse Thumpd2 (NM_028138.1) was constructed into plasmids pcDNA3.1(+)-Thumpd2. The copy number of standards could range from 101 to 1010, which based on the known concentrations of DNA standard molecules. RT-qPCR was performed using the standard curve method in QuantStudio 7 (Life Technology) with ChamQ Universal SYBR QPCR Master Mix as the dsDNA fluorescence dye. The amplification efficiency (E%) of standard material must be in the range of 90–110%. Thus, we designed several primers for each gene and pick up one that meets the amplification efficiency. The primers finally used for the Thumpd2 in the RT-qPCR are listed in Supplementary Table S1 .
10
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from tissues and cells with Trizol reagent (Invitrogen). RT-PCR was performed as described in the protocols (Invitrogen, Carlsbad, CA). The primers and conditions for RT-PCR analyses were as previously described (23), with the exception of NR4A2 (Nurr1) primers (Forward: 5′-AGA GAC GCG GAG AAC TCC TA-3′, Reverse: 5′-AGG CAT GGC TTC AGC CGA GT-3′). PCR was regularly amplified for 35 cycles, more or less, due to the difference in RNA abundance of these investigated genes. Primers for real-time PCR analyses were the same as described above. Real-time PCR was performed by using SYBR Green PCR master mix from QuantStudio™ 7 (Life technologies, USA). Properly diluted cDNA was used in a 10 μl qRT-PCR, in triplicate for each gene. β-actin was used as the internal control and results were calculated by applying 2−ΔΔCT methods. The experiments were done separately in triplicate.
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