Differential scanning fluorimetry was performed as previously described (Niesen). Breifly, proteins were diluted in PBS to a concentration of 100 μg/ml, and SYPRO Orange dye was added to a final concentration of 5×. Samples were exposed to a temperature gradient from 25–94°C with a 1 minute equilibration at each degree centigrade. Fluorescence was quantified in an Applied Biosystems ABI 7500 Fast Real-Time PCR machine.
Abi 7500 fast real time pcr machine
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 7500 Fast real-time PCR machine is a laboratory instrument designed for the amplification and detection of nucleic acid sequences using the real-time PCR method. It is capable of performing fast thermal cycling and provides accurate and reliable data for gene expression analysis, quantification, and genotyping applications.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Taqman primer probes sets, by Thermo Fisher Scientific (3 mentions)
Tripure, by Roche (3 mentions)
Qiasymphony sp platform, by Qiagen (2 mentions)
Virus blood 200 protocol, by Qiagen (2 mentions)
Fam zen iowa black quencher labeled taqman probe, by Thermo Fisher Scientific (2 mentions)
Nuclisens easymag platform, by bioMérieux (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Sybr protocols, by Takara Bio (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Mirnapure mini kit, by CWBIO (1 mentions)
Sybr green supermix, by Bio-Rad (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rnaeasy spin column, by Qiagen (1 mentions)
Dnase 1, by Promega (1 mentions)
34 protocols using abi 7500 fast real time pcr machine
1
Thermal Stability Profiling of Proteins
2
Quantitative PCR for HHV6b Detection
3
RNA Extraction and qPCR Analysis
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As described previously [11 ], cells were seeded at 0.5 million‧mL–1 and, 24 h later, were incubated with JDM‐7 for a further 48 h. Cells were then collected for RNA isolation. Two micrograms of total RNA from MV4‐11 were reverse transcribed into cDNA using Invitrogen Superscript II reverse transcriptase (Life Technologies) in accordance with the manufacturer's instructions. Random primers were used to obtain cDNA. Synthesized cDNA served as templates in 20‐µL quantitative PCR reactions. Quantitative PCR was performed using SYBR protocols (Takara, Dalian, China). The PCR was run in an ABI7500 fast real time PCR machine (Applied Biosystems, Foster City, CA, USA) with quantitative PCR cycling conditions of 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Relative concentrations of each target template were calculated according to the comparative Ct method. Expression of target transcripts was standardized to glyceraldehyde‐3 phosphate dehydrogenase. Quantitative PCR analyses were performed in triplicate. The variance between the groups is statistically similar. Three independent experiments were performed and P < 0.05 was considered statistically significant. The primers used are available upon request.
4
Quantitative PCR for HHV6b Detection
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Total nucleic acid was extracted from the whole blood using the QIAsymphony SP platform using the Virus Blood 200 protocol (Qiagen, Hilden, Germany) and cell preparations were extracted using the NucliSENS easyMAG platform (bioMerieux, Boxtel, the Netherlands) with final elution volume of 50 μL. For HHV6b detection, primers for HHV6 UL38 gene (forward [5’-GGA GTG CCT GTG GGT ATT C-3’], reverse [5’-CTA AGG TGA GCC AGA TTC G-3’]) and FAM/ZEN/Iowa Black quencher-labeled TaqMan probe were used (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed using an ABI 7500 Fast real-time PCR machine with standard cycling protocol (Applied Biosystems).
5
Quantification of sRNA Expression
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sRNAs were isolated from the cambial zone materials of 12 plants in LD3, SD8 and C5 using a miRNApure Mini Kit (CW Biotech, Beijing, China) following the manufacturer’s instructions. Then, the sRNA was polyadenylated by poly (A) polymerase, and first-strand cDNA was obtained from polyadenylated sRNAs using the miRNA cDNA Kit (CW Biotech, Beijing, China) following the manufacturer’s instructions. qRT-PCR was carried out as described: the SYBR Premix Ex Taq™ kit (TaKaRa Bio Inc., Japan) and an ABI 7500 Fast Real-time PCR machine (Applied Biosystems, Foster City, CA, USA) were used to complete the amplification, and the reaction procedure was set up according to the manufacturer’s protocol. Three replicates were performed for each sample with 5.8S rRNA as an internal reference [46 (link)], and we used the 2-ΔΔCT relative quantification method to calculate relative changes in gene expression [77 (link)]. All the primers are listed in Additional file 3 : Table S3.
6
Developmental Gene Expression Profiling
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Total RNA from each stage as well as from adults was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The quantity and quality of the RNA were assessed using agarose gel electrophoresis and a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA), respectively. Trace genomic DNA in the RNA solution was removed using the Turbo DNAfree Kit (Ambion Inc, Austin, TX). First-strand cDNA was synthesized from RNA using MMLV reverse transcriptase (USB, Cleveland, OH) with oligo dT(18) primer. Gene-specific primers were designed with Primer3 software [23] (link), and the sequences of all primers used are listed in Table S1 . According to the standard protocol, the qRT-PCR assays were conducted using SYBR Green Supermix (BioRad) on an ABI 7500 fast real-time PCR machine (Applied Biosystems, Foster City, CA). Cytochrome b (Cyb) was employed as an internal control for normalization [24] (link); the relative expression patterns were calculated based on the 2−ΔΔCt method [25] (link), [26] (link). Significant differences in expression patterns were analyzed by one-way ANOVA followed by the Tukey's post-hoc test.
7
Quantifying Viral Gene Expression by qRT-PCR
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RNA was isolated from 106 cells using RNAeasy spin columns as described by the manufacturer (Qiagen, Valencia, CA). Following isolation, total RNA was incubated with DNAse I (Promega, Madison, WI) and then amplified by qRT-PCR. IE, UL138 and GAPDH gene expression was determined using a Taqman Triplex qRT-PCR system (SIGMA, Poole, UK) with the following primers and probes: IE, 5′- CAA GAA CTC AGC CTT CCC TAA GAC and 5′- TGA GGC AAG TTC TCG AAT GC with the probe CCA ATG GCT GCA GTC AGG CCA TG -(TAM); UL138, 5′- CGC TGT TTC TCT GGT TAG and 5′- CAG ACG ATA CCG TTT CTC with the probe CCG ACG ACG AAG ACG ATG AAC -(Cy5); and GAPDH, 5′- GGA AGC TTG TCA TCA ATG and 5′- CCC CAC TTG ATT TTG GAG with the probe ATC ACC ATC TTC CAG GAG CGA G -(JOE). Reactions were set up using the Qiagen RT-PCR Quantitect kit (Qiagen, Sussex, UK) in accordance with the manufacturer's protocol and the samples amplified for ABI 7500 Fast Real Time PCR machine (Applied Biosystems, Foster City, CA; 95°C for 15 s and 60°C for 45 s).
8
Quantitative gene expression analysis in fish
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Gonads and head-kidneys were collected from wild-type and cyp17a2-/- XY fish (n≥3 fish/genotype). Total RNA was isolated for each sample using RNAiso Plus (Takara, Dalian, China), and reverse transcribed using the Prime Script RT Master Mix Perfect Real Time Kit according to the manufacturer’s instructions (Takara, Dalian, China). All qRT-PCRs were carried out in an ABI-7500 fast Real-time PCR machine (Applied Biosystems, USA), and all experiments were performed according to the manufacturer’s instructions. The relative abundance of target genes was normalized to β-actin using R=2-ΔΔCt formula (30 (link)). The primer sequences used for the PCR reactions were listed in Table 1
9
Quantitative Analysis of Gene Expression in Murine Hearts
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Total RNA was extracted from the heart of wild-type and TRIP-Br1−/− mice by using an RNA extraction kit (Takara Minibest universal RNA extraction kit), and cDNA was synthesized using Superscript II reverse transcriptase from Invitrogen. Quantitative PCR (qPCR) amplifications of various genes were performed using SYBR Green (Takara) and an ABI 7500 fast real-time PCR machine (Applied Biosystems); GAPDH was used as a normalization control. After initial denaturation (30 s, 95°C), amplification was performed using 40 cycles of 5 s at 95°C and 34 s at 60°C. All qPCR data represent means ± SEM. The sequences of the primers used for qPCR are listed below:
AC1: 5′-CCTCGCACTTACTGGTCACA-3′ and 5′-AACCCACGATGTCTGCAAAC-3′;
GAPDH: 5′-CTTGTCATCAACGGGAAGCC-3′ and 5′-CATGAGCCCTTCCACAATGC-3′.
AC1: 5′-CCTCGCACTTACTGGTCACA-3′ and 5′-AACCCACGATGTCTGCAAAC-3′;
GAPDH: 5′-CTTGTCATCAACGGGAAGCC-3′ and 5′-CATGAGCCCTTCCACAATGC-3′.
10
Quantifying Mammary Gland Gene Expression
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Total RNA was isolated from 50–100 mg of mammary gland tissue using TRIzol (Life Technologies, Grand Island, NY) according to the manufacturer's protocols. The RNA samples were reverse-transcribed using the PrimeScript™RT reagent Kit with gDNA Eraser (Takara Bio, Dalian, China). The quality of RNA samples was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). All samples had an RNA integrity number factor >7.3. β-actin was used as the internal control gene to normalize the mRNA expression. Primers were designed and synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China) based on the sequences reported in GenBank. The primer sequences used in this study are listed in Table 2 . qRT-PCR was carried out using Novostart SYBR qPCR SuperMix Plus (Novoprotein, Fremont, CA) in an ABI 7500 fast Real-Time PCR machine (Applied Biosystems, Waltham, MA) following the manufacturer's instructions. cDNA from 1 μg total RNA was used as the template; the following cycling conditions were used: 95°C for 1 min for initial denaturation and enzyme activation, 40 cycles of 95°C for 20 s and 60°C for 1 min for amplification. The 2−ΔΔCT method was used to calculate the relative gene expression with data from samples collected at enrolment (baseline sample) as calibrator samples.
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