Total RNA was extracted from skeletal muscle samples using AGPC methods, and was used as templates for cDNA synthesis with ReverTra Ace qPCR RT Master Mix and gDNA Remover (Toyobo). RT-qPCR reactions were performed using Prism 7900HT or Step One Plus Real-Time PCR Systems (Thermo Fisher) with AmpliTaq Gold® 360 Master Mix (Thermo Fisher), EvaGreen (Biotium), and the primers listed in Table 1 . Ribosomal phosphoprotein P0 (Rplp0/36B4) mRNA was used as an internal control.
Prism 7900ht
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Prism 7900HT is a real-time PCR instrument designed for high-throughput nucleic acid analysis. It features a high-density sample capacity, rapid thermal cycling, and advanced data analysis software. The core function of the Prism 7900HT is to perform accurate and sensitive real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (18 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (5 mentions)
First strand synthesis system, by Thermo Fisher Scientific (5 mentions)
Dneasy blood tissue kit, by Qiagen (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Sds 2, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Amplitaq gold 360 master mix, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Gdna remover, by Toyobo (2 mentions)
Evagreen, by Biotium (2 mentions)
Revertra ace qpcr rt master mix, by Toyobo (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Taqman primers and probe, by Thermo Fisher Scientific (2 mentions)
Tmsb4, by Merck Group (2 mentions)
Gotaq qpcr master mix, by Promega (2 mentions)
Sybr green supermix kit, by Toyobo (2 mentions)
Trizol reagent, by Merck Group (2 mentions)
73 protocols using prism 7900ht
1
Skeletal Muscle RNA Extraction and RT-qPCR
2
Transcriptome Analysis of Tissue Samples
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Total RNA was extracted from tissue or cell samples using AGPC methods and was used as a template for cDNA synthesis with the ReverTra Ace qPCR RT Master Mix and gDNA Remover (Toyobo). qPCR was performed using Prism 7900HT or Step One Plus Real-Time PCR Systems (Thermo Fisher) with AmpliTaq Gold® 360 Master Mix (Thermo Fisher), EvaGreen (Biotium), and the primers listed in Table S1 ; 18S rRNA served as an internal control. Prior to the microarray analysis, a Low Input Quick Amp Labeling Kit (Agilent Technologies) was used to label total RNA that had been purified using the RNeasy MinElute Cleanup Kit (Qiagen). The labeled RNA was then used to probe a SurePrint G3 Mouse Gene Expression 8 × 60K Microarray (Agilent Technologies), and the signals were scanned using a G2565 microarray scanner (Agilent Technologies). Microarray data were extracted from the scanned images using Feature Extraction 10.7 software (Agilent Technologies), and the raw unfiltered microarray data were deposited in the Gene Expression Omnibus dataset (subseries entries GSE149423 ). The differentially expressed genes were isolated and analyzed using the Subio Platform (Subio).
3
Quantification of Gene Expression via qRT-PCR
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Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer’s instructions. Then, RNA was transformed to cDNA with reverse transcription using a PrimeScript II RT Reagent Kit (Takara, Kyoto, Japan). Quantification of specific gene expression was measured with SYBR premix Ex Taq™ (Takara) and a Prism 7900HT sequence detection system (Thermo Fisher Scientific, Rockford, IL, USA) following the manufacturer’s protocols. Relative gene expression was determined using the 2ΔCt (internal control) − ΔCt (gene) method and normalized to cyclophilin [53 (link)]. Data was acquired from at least triplicate experiments and denoted as mean ± standard deviation (SD). Used primer sequences are listed in Supplementary Table S2 .
4
KLF2 and S1P1 Expression in CD8+ T Cells
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At day 30 after x31 infection, CD69+ or CD69− fractions of CD44hiCD8+ T cells in each tissue were sorted on a FACSAria flow cytometer (BD). Total RNA was prepared using an RNeasy Plus Mini kit (QIAGEN), and cDNA synthesis was performed by using a PrimeScript RT reagent kit (Takara Bio Inc.). Quantitative PCR was done on a real-time PCR system (Prism 7900HT; Thermo Fisher Scientific) in a 96-well plate format with SYBR green–based detection (Takara Bio Inc.). β-actin mRNA was used for normalization. The primers used were: KLF2 forward, 5′-TGTGAGAAATGCCTTTGAGTTTACTG-3′ and KLF2 reverse, 5′-CCCTTATAGAAATACAATCGGTCATAGTC-3′; and S1P1 forward, 5′-GTGTAGACCCAGAGTCCTGCG-3′ and S1P1 reverse, 5′-AGCTTTTCCTTGGCTGGAGAG-3′. mRNA levels in the thymocytes were used as a standard, and relative expression of mRNA was calculated as 2−ΔCt.
5
RNA Extraction, Reverse Transcription, and RT-PCR
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RNA was purified from tissue samples using the RNeasy Mini Kit following the manufacturer's instructions as described previously (Qiagen, UK; Cui et al., 2016 ; Li, Guabiraba, et al., 2014 ). Reverse transcription (RT) of RNA into cDNA was carried out using High‐Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific, MA, USA). Real‐time polymerase chain reaction (RT‐PCR) was performed using Fast SYBR Green master mix on a Prism 7900HT (Thermo Fisher Scientific). The primers used were as follows: Il4, forward 5′‐CAT GGC TTG GGT ACA GGT CT‐3′, reverse 5′‐TTT GTA GTG GGA GGG GAC AG‐3′; Il5, forward 5′‐GAA GTG TGG CGA GGA GAG AC‐3′, reverse 5′‐GCA CAG TTT TGT GGG GTT TT‐3′; Il13, forward 5′‐GAA TCC AGG GCT ACA CAG AAC‐3′, reverse 5′‐AAC ATC ACA CAA GAC CAG ACT C‐3′; Ifng, forward 5′‐ACT TTG CTT CTG CCT TTC CA‐3′, reverse 5′‐ACA AGG TCA CCC ACA GGA‐3′.
6
Quantifying IL-20 Signaling Pathway
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RNA was isolated from the treated Min6 β-cells and mouse islets or untreated db/db mouse islets using the RNeasy kit with the QiaShredder column (Qiagen, Denmark) following the manufacturer’s protocol. RNA was reverse transcribed to cDNA using cDNA Archive kit (Life Technologies, Denmark) according to the manufacturer’s protocol. qRT-PCR was performed on the Applied Biosystems Prism 7900HT real-time PCR machine and analyzed using SDS 2.4 software (Life Technologies, Denmark). The following primer/probes were purchased from Life Technologies: IL20 (Mm00445341_m1), IL20Ra (Mm00555504_m1), IL20Rb (Mm01232398-m1), IL22R (Mm00663697_m1), SOCS3 (Mm00545913_s1) and Rn18S (Hs99999901_s1). Relative transcript quantities were calculated by the standard curve method and normalized to the reference gene Rn18S.
7
Quantitative RT-PCR of Ackr2 Expression
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RNA was extracted using RNeasy columns with DNAse treatment (Qiagen, Manchester, UK), complementary DNA generated (AffinityScript (Agilent, Santa Clara, CA, USA)) and quantitative PCR done using Ackr2-specific Taqman assay (mm0044555_m1) (Life Technologies, Paisley, UK) on a Prism 7900HT (Life Technologies). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probes were used to normalize Ackr2 expression. Analysis used the relative quantitation ΔΔ−2 CT method to give the relative quantification (fold change) value, with naive tissue as calibrators set to 1.
8
Quantifying P2RX7 Expression in CXCR5+ CD4 Cells
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CXCR5+ CD4 cells were sorted on a FACSAria (BD) from PBMCs. RNA was extracted using the RNeasy Mini Kit (Qiagen) and converted to cDNA. Random primers and Moloney murine leukemia virus reverse transcription (Invitrogen) were used for cDNA synthesis. Transcripts were quantified by real-time quantitative PCR on an ABI PRISM 7900HT with Applied Biosystem predesigned TaqMan gene expression assays and reagents according to the manufacturer’s instructions. The following probe was used (identified by Applied Biosystem assay identification number): P2RX7 (Hs00175721_m1). For each sample, mRNA abundance was normalized to the amount of TAT-box binding protein (Hs00427620_m1), used as gene reference, and expressed as arbitrary units.
9
Gene Expression Analysis of HLI in Hamstring
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Total RNA was isolated from hamstring muscle distal to the site of surgery following 3 days of HLI and sham surgeries using Qiagen Fibrous Tissue RNA isolation kit. Expression of the genes encoding Atpgd1, Cndp2, Pht1, TauT, Vegf, and Pept2 were determined using quantitative RT-PCR and primers described previously by Everaert et al. (2013a) (link) (Table 1 ). Results were normalized to HPRT1 and expressed according to the comparative Ct method that compares the Ct values of the gene of interest and sham as an internal control gene (Livak and Schmittgen, 2001 (link)). Measurements were made using Prism 7900 HT (Applied Biosystem).
10
Cytokine Expression Analysis by qRT-PCR
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Total RNA was extracted with TRIzol reagent and mRNA was transcribed with random hexamers using Super Script II (Invitrogen). SYBR-green Premix Ex-Tag II (Takara, Madison, WI) was used for the quantification of cytokine transcripts with a real-time quantitative PCR on an Applied Biosystem Prism 7900HT sequence detection system. PCR results were analyzed using the comparative ddCt method with cyclophilin as control. The experiments were performed in triplicates and expressed as the mean S.D. The p values were determined with a two-tailed t-test.
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