Spheroids of the 105 strain were prepared as described previously, randomly split into five groups, and incubated in media with different sucrose content (0, 10, 30, 50, and 70 mM) at RT. Every 6 hours, a sample of 10 spheroids was randomly taken from each of the conditions, lysed in 350 μl of RL buffer with 1% β-mercaptoethanol (Single Cell RNA Purification Kit, Norgen), frozen on dry ice, and stored at −80°C. After collecting all the samples, RNA was isolated as outlined in the instructions for the kit. The concentration and purity of the extracted RNA was verified using NanoDrop 1000 (Thermo Fisher Scientific). Next, cDNA was prepared with the Oligo(dT)12-18 Primer (Thermo Fisher Scientific) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) as per manufacturer’s instructions. The qPCR reactions were carried out using the StepOnePlus Real-Time PCR cycler (Thermo Fisher Scientific) with a standard run method. Each reaction contained 10 ng of cDNA, 1× the Platinum SYBR Green qPCR SuperMix-UDG w/ROX (Invitrogen) master mix with 0.25 μM primers in a total volume of 25 μl. The quantification of gene expression was performed using the StepOne software v 2.3 (Thermo Fisher Scientific). Table 2 below shows the sequences of primers used.
Platinum sybr green qpcr supermix udg w rox
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Platinum SYBR Green qPCR SuperMix-UDG w/ROX is a ready-to-use solution for quantitative real-time PCR (qPCR) applications. It contains all the necessary components, including SYBR Green I dye, Platinum Taq DNA polymerase, dNTPs, and a passive reference dye (ROX).
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Oligo dt 12 18 primer, by Thermo Fisher Scientific (3 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (3 mentions)
Stepone software v2, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr cycler, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Viia7 qpcr detection system, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Mmlv reverse transcriptase 1st strand cdna synthesis kit, by Illumina (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Mlv rt kit, by Thermo Fisher Scientific (1 mentions)
Total rna kit 1, by Omega Bio-Tek (1 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (1 mentions)
Superscript 3 first strand synthesis supermix for qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
18 protocols using platinum sybr green qpcr supermix udg w rox
1
Sucrose Impact on Cell Spheroid Transcriptome
2
Quantitative Real-Time PCR for B7-H6 Expression
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Cells were split and grown in equal densities for at least 24 hours prior to lysis. Cells were lysed and RNA was isolated using the Quick RNA Miniprep kit (Zymo). cDNA was prepared from 0.25–1 μg total RNA using mMLV reverse-transcriptase (Invitrogen) and oligoDT primers. The cDNA was mixed with 3 μM of each of the primers (forward and reverse) and 5 μl of Platinum SYBR Green qPCR SuperMix-UDG w/ROX (Invitrogen) was added. B7-H6 was compared between samples, HCMV US9 was measured as a control for infection levels, and hUBC together with hHPRT were used as endogenous reference genes. The quantitative real-time PCR reaction took place on a QuantStudio12K Flex Real Time PCR System (Applied Biosystems).
3
Quantitative miRNA expression analysis
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Reverse transcription (RT) of target miRNAs was performed using gene-specific RT primers and MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (Epicentre Biotechnologies, Madison, WI, USA) according to manufacturer’s instructions. Relative qRT-PCR of individual miRNA was then performed using Platinum SYBR Green qPCR SuperMix-UDG w/ROX (Invitrogen). Expression levels of target miRNAs were normalized to the small RNA U6 internal control. Primers used were listed in S1 Table . All experiments were performed in triplicate.
4
Foot Regeneration Transcription Factor Knockdown
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Knockdowns of foot-specific transcription factors and bisections were performed as described above. On the third day after the bisection, three foot-regenerating halves were collected per sample and lysed in 350 μl of RL buffer with 1% β-mercaptoethanol (Single Cell RNA Purification Kit, Norgen), frozen on dry ice, and stored at −80°C. After collecting all the samples, RNA was isolated as outlined in the instructions for the kit. The concentration and purity of the extracted RNA was verified using NanoDrop 1000 (Thermo Fisher Scientific). Next, cDNA was prepared with the Oligo(dT)12-18 Primer (Thermo Fisher Scientific) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) as per manufacturer’s instructions. The qPCR reactions were carried out using the StepOnePlus Real-Time PCR cycler (Thermo Fisher Scientific) with a standard run method. Each reaction contained 10 ng of cDNA, 1× the Platinum SYBR Green qPCR SuperMix-UDG w/ROX (Invitrogen) master mix with 0.25 μM primers in a total volume of 25 μl. The sequences of the primer pairs used are given in Table 1 below. The quantification of gene expression was performed using the StepOne software v 2.3 (Thermo Fisher Scientific).
5
Analyzing Wnt Ligand Expression in Muscle Fibers
6
Quantifying ADAR Isoform Expression in Mice
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Two hundred and fifty nanograms of RNA were used in a reverse transcription reaction using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Cat. No. AB 1453). The cDNA obtained was used in real‐time PCR. Platinum SYBR GreenqPCR SuperMix-UDG w/ROX (Invitrogen, Carlsbad, CA, USA) was used to quantify gene expression. The number of mice used for this experiment was n = 7 for the control mice and n = 5 for the PolyI:C group, since not enough DNA was available from this group. Specific primers were used to amplify ADAR-p110 and ADAR-p150, and β-actin was included in each experiment as a loading control. The fold change in expression was determined using the ΔCt method. The following primer sequences were used for PCR—ADAR-p110 Forward Primer (FP): 5′-GCAGCGTCCGAGGAATCG-3′, Reverse Primer (RP): 5′-TAAGACTCCGGCCCCTGTG-3′; ADAR-p150 FP: 5′-CACTATGTCTCAAGGGTTCAGGG-3′, ADAR-p150 RP: 5′-CACTTGCTATGCTCATGACTAGGG-3′; and β-actin FP: 5′-AGAGCATAGCCCTCGTAGAT-3′, β-actin RP: 5′-CCCAGAGCAAGAGAGGTATC-3′.
7
Quantitative PCR Analysis of Immune Regulators
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Total RNA was extracted by E.Z.N.A. Total RNA kit I (Omega Bio-Tek, Inc., Norcross, GA, USA), and cDNA synthesis with MLV RT kit (Invitrogen) for 50 min at 37°C in the presence of oligo-dT primer. qPCR analyses were performed by Platinum® SYBR®-Green qPCR SuperMix-UDG w⁄ROX (Invitrogen Life Technologies, Carlsbad, CA, USA) on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems Life Technologies, Foster City, CA, USA). Primers used were listed as follows: β-actin forward: 5′-CAGAGCAAGAGAGGCATCC-3′, and reverse: 5′-CTGGGGTGTTGAAGGTCTC-3′; IL-9 forward: 5′-CCAGCTTCCAAGTGCCACTGC-3′, and reverse: 5′-TGCATGGTGGTATTGGTCATCTG-3′; BATF forward: 5′-ACACAGAAGGCCGACACC-3′, and reverse: 5′-CTTGATCTCCTTGCGTAGAGC-3′; IRF4 forward: 5′-ACCCGCAGATGTCCATGAG-3′, and reverse: 5′-GTGGCATCATGTAGTTGTGAACCT-3′; PU.1 forward: 5′-GAAGACCTGGTGCCCTATGA-3′, and reverse: 5′-CTCTGGAGCTCCGTGAAGTT-3′; IL-10 forward: 5′-GCCTAACATGCTTCGAGATC-3′, and reverse: 5′-TGATGTCTGGGTCTTGGTTC-3′.
8
Total RNA Extraction and Real-Time qPCR Analysis
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Total RNA was purified using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and ZR RNA MicroPrep (Zymo Research, USA). After concentration and integrity validation (NanoDrop 1000, Thermo Fisher Scientific, USA), cDNA was generated using 0.5–1 μg of RNA with SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen, USA). Real-Time qPCR was performed in technical triplicates on a ViiA™ 7 Real-Time PCR System with 384-well plate (Applied Biosystems, USA) with a Platinum® SYBR® Green qPCR SuperMix-UDG w/ROX (Invitrogen, CA, USA) and primers mix at final concentration of 250 nM. Gene expression (Cycle threshold) values were normalized based on the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) housekeeping gene and the Delta CT calculated.
9
Quantitative Analysis of Immune Genes
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1 μg RNA from each sample was reverse transcribed into cDNA by SuperScript® III First-Strand Synthesis SuperMix (Invitrogen, Life Technologies) following the manufacturer’s instructions. qRT- PCR was performed using Platinum® SYBR® Green qPCR SuperMix-UDG w/ROX (Invitrogen, Life Technologies). Relative expression of CSF2, BIRC3, IL8, ITGAV, IFI27, MMP1 and CSH1 mRNAs were normalized to four housekeeping genes: UBC, RPLP0, ACTB and PPIA. The sequences of the qRT-PCR primer pairs for each gene are shown in Supplementary Table 4 .
10
Quantitative Real-Time PCR Analysis of C2C12 Cells
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For C2C12 mouse myoblasts, equal amounts of DNA (0.5 ng) were used for real-time PCR with Platinum SYBR Green qPCR SuperMix-UDG w/ROX (Invitrogen, Paisley PA4 9RF, UK) on a StepOne-Plus Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) according to the manufacturer’s instruction. UDG was inactivated for 2 min at 50 °C and DNA was denatured for 10 min at 95 °C. Cycle parameters were set to 40 cycles of 15 s at 95 °C and 45 s at 60 °C. Specificity of amplification products was confirmed by melting curve analysis. DNA levels were normalized to Gapdh and calculated using the comparative CT method. Primers for quantitative real-time PCR contained the following sequences: Gapdh forward: 5-CCA TACATACAGGTT TCT CCA G-3, Gapdh reverse: 5-CTG GAA AGCTGT GGC GTG ATG G-3,MajSat forward: 5-GGC GAG AAA ACT GAA AAT CAC G-3, MajSat reverse (20): 5-AGG TCC TTC AGT GTG CAT TTC-3108 (link).
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