RNA was extracted using TRIzolTM (Thermo Fisher Scientific, #15596026) or RNeasy Mini Kit (Qiagen, #74104) and reverse transcribed using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific, #K1691) according to the manufacturers’ instructions. Quantitative PCR (qPCR) was performed using TaqMan Universal PCR Mastermix (Thermo Fisher Scientific, #4304437) and TaqMan probes for PI3KR1, AKT3, HK, Glut4, Top1mt, and Pparg or PrimeTime Std qPCR Assay (Integrated DNA Technologies) for Infg and Tgfb1(Supplementary Table 3 ). Gene expression was normalized to GAPDH and Gapdh or b2m (Thermo Fisher Scientific).
Primetime std qpcr assay
Manufactured by Integrated DNA Technologies
Sourced in United States, Belgium
The PrimeTime Std qPCR Assay is a pre-designed, dual-labeled DNA probe-based quantitative PCR (qPCR) assay developed by Integrated DNA Technologies. The assay is designed to quantify specific DNA targets in real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Cfx96 c1000 qpcr machine, by Bio-Rad (2 mentions)
Tissuelyser 2 equipment, by Qiagen (2 mentions)
Ribopure kit, by Thermo Fisher Scientific (2 mentions)
Taqman array mouse immune response, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Thermo Fisher Scientific (2 mentions)
Revertaid rt reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (1 mentions)
Rnasin, by Avantor (1 mentions)
Sensifast probe mix, by Meridian Bioscience (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Qx200 system, by Bio-Rad (1 mentions)
One step rt ddpcr advanced kit for probe, by Bio-Rad (1 mentions)
Power sybr green cells to ct kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
16 protocols using primetime std qpcr assay
1
Quantitative Analysis of Metabolic Genes
2
Gene Expression Analysis of PCNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with RNA Cell Lysis Buffer (Boston Bioproducts, R-108) supplemented with 1:40 dilution of RNasin (VWR, PAN2611) and incubated for 5 minutes at room temperature. qRT-PCR reaction was prepared using TaqMan Fast Virus 1-Step Master Mix (Life Technologies, 4444436) according to manufacturer's protocol. For a final volume of 10 μL, 1 μL of template was added to the 384-well plate (Applied Biosystems, 4309849). A PrimeTime Std qPCR Assay was used to detect PCNA with a 6-FAM/ZEN/IBFQ 5′-3′ quencher (Integrated DNA Technologies, 326107661). Human RPLPO-VIC/MGB (Life Technologies, 4326314E) was used for normalization and was plexed with PCNA probe for all samples. Reaction was performed using QuantStudio 6 Flex.
3
Methylation Analysis of IL-6 Promoter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The promoter sequences of IL-6, and Alu as the reference gene, were obtained from the Ensembl genome browser (www.ensembl.org ). The forward and reverse primers, and probes were designed using the MethPrimer software tool (http://www.urogene.org/methprimer/ ) and procured as PrimeTime® Std qPCR Assay (Integrated DNA Technologies, USA). Sequence of primers and probes used in the MethyLight assay were:
The assay efficiency was determined prior to running the assay according to method described in a previous study (18 (link)).
For every sample, 25 ng bisulphite-converted DNA was added to 1x SensiFAST™ PROBE mix (Bioline, UK) and 1x PrimeTime® Std qPCR Assay in a 10 mL polymerase chain reaction (PCR) using Bio-Rad CFX96™ Real-Time System (Bio-Rad, USA). IL-6 and Alu were assayed in the same 96-wells plate. PCR reaction condition was 95 °C for 10 min (polymerase activation), followed by 95 °C for 10 s (denaturation) and 60 °C for 30 s (annealing and extension) and the cycle was repeated for 60 cycles. The IL-6 methylation of each sample was assessed as .
The assay efficiency was determined prior to running the assay according to method described in a previous study (18 (link)).
For every sample, 25 ng bisulphite-converted DNA was added to 1x SensiFAST™ PROBE mix (Bioline, UK) and 1x PrimeTime® Std qPCR Assay in a 10 mL polymerase chain reaction (PCR) using Bio-Rad CFX96™ Real-Time System (Bio-Rad, USA). IL-6 and Alu were assayed in the same 96-wells plate. PCR reaction condition was 95 °C for 10 min (polymerase activation), followed by 95 °C for 10 s (denaturation) and 60 °C for 30 s (annealing and extension) and the cycle was repeated for 60 cycles. The IL-6 methylation of each sample was assessed as .
4
Transcriptional Profiling of Targeted Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from the PB using the RNeasy Mini Kit (QIAGEN). Transcription levels of TRAP1, GPX1, and RAP1B in Pt1 and DPP4, PERP, TNFAIP3, MLLT10, and SMARCC1 in Pt2 and the reference gene TBP were analyzed using the Prime Time Std qPCR Assay (Integrated DNA Technologies, Coralville, IA, USA) and One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad), followed by the calculation of signal-positive droplets using the Bio-Rad QX200 system. The expression level of each gene was normalized relative to the expression of TBP.
5
Quantitative RT-PCR for TGDF1 and ACTB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse and human TDGF1 (forward: TCCTTTTGTGCCTGCCCTC; reverse: CACAGGGAACACTTCTTGG) or commercially available primers for ACTB (Integrated DNA Technologies, Coralville, IA, IDT-PrimeTime Std qPCR Assay Hs.PT.39a.22214847) were included in standard PCR or quantitative real-time RT-PCR using the Power SYBR Green Cells-to-CT kit (Thermo Fisher Scientific, Waltham, MA, #4402954) according to the manufacturer’s instructions. Real-time PCR was carried out on a lightCycler 480 (Roche, Basel, Switzerland), and comparisons were made using the ΔΔCt method with β-actin as a control transcript.
6
Quantitative mtDNA Analysis using qPCR
7
Quantifying Gene Expression by qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from cultured cells using the RNeasy Mini Kit (Qiagen, Mississauga, Canada), according to the manufacturer's instructions. RNA was stored in RNase-free water at −80 °C until reverse transcription. Following the manufacturer's instructions, we synthesized the first-strand cDNA from 1 μg total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). Target mRNA expression levels were measured using Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following PrimeTime® Std qPCR Assay (Integrated DNA Technologies, Coralville, Iowa, USA): GAPDH (assay ID Hs.PT.39a.22214836), NFE2L2 (assay ID Hs.PT.58.28159373), and HMOX1 (assay ID Hs.PT.58.45340055). All reactions were performed in triplicate. The housekeeping gene, GAPDH, was used for normalization. Comparative CT method was used to calculate the relative amounts of mRNA.
8
Quantification of Gene Expression in HCV-infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from HCV-infected and non-infected Huh7.5 cells using the RNeasy kit (Qiagen). Equal amounts of total RNA were reverse transcribed using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Oligonucleotides were designed using the Primer3 PCR Primer Design Tool. The qRT-PCR analysis was performed using the Power SYBR Green Master Mix (Life Technologies, Carlsbad, CA, USA) and gene sequence-specific primers (Table S1 ).
Alternatively, genes were amplified using the gene-specific PrimeTime® Std qPCR Assay (Integrated DNA Technologies, Coralville, IA, USA). The thermal program included 10 min incubation at 95 °C followed by 40 cycles of 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 20 s. The fluorescence readings were recorded after each 72 °C step. Dissociation curves were performed after each PCR run to ensure that a single PCR product had been amplified. Differential expression was calculated using the equation: 2(−ΔΔCt), with GAPDH as a housekeeping gene control.
Alternatively, genes were amplified using the gene-specific PrimeTime® Std qPCR Assay (Integrated DNA Technologies, Coralville, IA, USA). The thermal program included 10 min incubation at 95 °C followed by 40 cycles of 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 20 s. The fluorescence readings were recorded after each 72 °C step. Dissociation curves were performed after each PCR run to ensure that a single PCR product had been amplified. Differential expression was calculated using the equation: 2(−ΔΔCt), with GAPDH as a housekeeping gene control.
9
Quantitative Analysis of mtDNA Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the total levels of mtDNA present in samples, we performed qPCR using TaqMan reagents (PrimeTime Std qPCR Assay, Integrated DNA Technologies)20 (link). Samples were run on a BioRad CFX96/C1000 qPCR machine. Comparative cycle threshold (Ct) method was used to determine relative reads and total mtDNA levels were determined by comparing mtDNA (ND1 and ND5) to genomic DNA (18S). The following primer/probe sets were used20 (link):
10
Quantification of PTPN5 mRNA in Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the striatum and hippocampus of BDNF +/+ and BDNF -/-mice at P7 using the Total RNA Isolation Nucleospin RNA II Kit (Macherey-Nagel, Düren, Germany). Purified RNA (500 ng) was reverse transcribed using the PrimeScript™ RT reagent Kit (Perfect Real Time; Takara Biotechnology (DALIAN) CO., LTD, Japan). The cDNA synthesis was performed at 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 min in a final volume of 20 μl. The cDNA was then analyzed by quantitative real-time-PCR using a TaqMan® Gene Expression Assay (Applied Biosystems, Foster City, CA) for PTPN5 (Mm00479063_m1). Q-PCR was performed in a final volume of 12.5 μl using the Premix Ex Taq™ (Probe qPCR) (Takara Bio INC). Reactions included 40 cycles of a two-step PCR: 95 °C for 5 s and 60 °C for 20 s, after initial denaturation at 95 °C for 30 s. All Q-PCR assays were performed in duplicate. To provide negative controls, and exclude contamination by genomic DNA, the reverse transcriptase was omitted in the cDNA synthesis step. The data were analyzed and quantified using the Comparative Quantitation Analysis program of the MxProTM Q-PCR analysis software version 3.0 (Stratagene) with the 18S gene expression (PrimeTime Std qPCR Assay; Integrated DNA Technologies; Coralville, IA) as internal loading control. Results were expressed relative to wild-type values.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!