Tumor cell lines were treated with mouse IFN-γ (Peprotech, cat 315-05) and TNF-α (R&D systems; catalog 410-MT-010) at 100 ng/ml for 8 hours. Tumor cells were treated with or without CX3CR1 antibody (10 µg/ml) for 30 minutes at 37°C followed by CX3CL1 (Novus Biologicals; cat NBP2-35038) at 2 µg/ml for 8 hours. RNA was isolated using RNeasy kit (Qiagen catalog 74104) using manufacturer’s protocol and cDNA was made using Bio-Rad cDNA kits. mRNA was quantified using Taqman qPCR (Applied Biosystems, catalog 4304437). The assay was run on Applied Biosystems Quant Studio 6 real time PCR system. Expression levels were normalized to GAPDH. Primers were purchased from Applied Biosystems; mouse PD-L1 (Mm03048248_m1), mouse IL-6 (Mm00446190_m1), mouse NLRP3 (Mm00840904_m1) and mouse GAPDH (Mm99999915_g1).
Taqman qpcr
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan qPCR is a real-time PCR technology used for gene expression analysis and nucleic acid quantification. It utilizes fluorescent TaqMan probes to detect and measure the amplification of target DNA sequences during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy kit, by Qiagen (5 mentions)
Steponeplus, by Thermo Fisher Scientific (5 mentions)
Sybr green, by Thermo Fisher Scientific (5 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Stepone v2, by Thermo Fisher Scientific (3 mentions)
Universal probe library, by Roche (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Rneasy kit, by Qiagen (2 mentions)
Superscript 2 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (2 mentions)
Cyclophilin assay rn 00690933 m1, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (2 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Proteinase k, by Merck Group (2 mentions)
Dnase 1, by New England Biolabs (2 mentions)
43 protocols using taqman qpcr
1
Cytokine-Induced Immune Checkpoint Regulation
2
Quantifying DNMT1 Expression via qPCR
3
Quantifying PPAR-γ Expression in Adipose Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from epididymal adipose tissue using the reagent TRIzol (Invitrogen). The SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen) kit was used for the synthesis of 20 μl of complementary DNA from 1000 ng of total RNA. The mRNA levels of PPAR-γ (Rn00440945_m1, Applied Biosystems) was determined by real-time PCR. Quantitative measurements were made with a commercial kit (TaqMan qPCR; Applied Biosystems) in a detection system (StepOne Plus; Applied Biosystems). Cycling conditions were as follows: enzyme activation at 50 °C for 2 min, denaturation at 95 °C for 10 min, complementary DNA products were amplified for forty cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Gene expression was quantified in relation to the values of the C group after normalization by an internal control (cyclophilin: assay Rn 00690933_m1; Applied Biosystems) by the method 2-ΔΔC T, as described previously (Livak and Schmittgen, 2001 (link)).
4
Quantification of miRNA in Blood Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples from experiment 1 were used to verify the presence of miRNA by the use of TaqMan-qPCR (Applied Biosystems). miRNA was detected by running quantitative real-time PCR (qRT-PCR) on all samples isolated from the volunteers. A serial dilution of cervical adenocarcinoma (HeLa-S3) total RNA (Ambion Life Technologies, cat. Nr: AM7852) was used to make a standard curve (range: 200 to 0.02 ng/µL). Total RNA (40 ng) was reverse transcribed in a 15 µL reaction using TaqMan reverse transcription reagents (Applied Biosystems). The TaqMan MicroRNA Assay IDs 000391, 001098, and 002626 (Applied Biosystems) were used to quantify the expression of hsa-miR-16, hsa-miR-181, and hsa-miR-423, respectively. Quantitative PCR was carried out on a StepOnePlus Real-Time PCR System (Applied Biosystem). The concentration (ng/µL) of miR-16, miR-181 and miR-423 in the blood samples was calculated from the standard curve equation.
5
Quantifying RAGE mRNA Expression in Kidney
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the kidney using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The SuperScript II First-Strand Synthesis System for real-time PCR (Invitrogen) kit was utilized for the synthesis of 20 μl of complementary DNA from 1000 ng of total RNA. The mRNA levels of RAGE (Applied Biosystems, Thermo Fisher Scientific Inc., Foster City, CA, USA) were determined by real-time PCR. Quantitative measurements were made using a commercial kit (TaqMan qPCR; Applied Biosystems) in a detection system (StepOne Plus; Applied Biosystems). Cycling conditions were as follows: enzyme activation at 50 °C for 2 min, denaturation at 95 °C for 10 min, complementary DNA products amplification for 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Gene expression was quantified in relation to the values of the control group after normalization to an internal control (cyclophilin; Applied Biosystems).
6
RNA Extraction and qPCR Analysis
7
Quantifying Cardiac Marker Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from undifferentiated hPSCs and derived CMs at day 14 of differentiation using RNeasy Mini Kit (Qiagen). Synthesis of cDNA was carried out using 1 μg RNA with SuperScript III Reverse Transcriptase Kit (Invitrogen), according to manufacturer's instructions. ADRB2 analysis was with TaqMan qPCR (No. Hs00240532_s1; Applied Biosystems) and signals were normalized to GAPDH (No. Hs99999905_m1; Applied Biosystems) as the housekeeping gene, following the manufacturer's instructions. Semi-qPCR cycle conditions were 95°C for 2 min, 64.5°C for 30 s (GRK5, ACTC1, RYR2, and ACTB), and 72°C for 60 s, with a final elongation step of 72°C for 10 min. Each reaction used 250 ng of cDNA with Phusion polymerase (NEB) for 35 cycles. Gels were imaged with a LAS-4000 (Fujifilm) image analyzer, densitometry was carried out using FIJI, and a version of ImageJ (National Institutes of Health) and signals were normalized to ACTB as the housekeeping gene. Primers for expression analysis are provided in Supplementary Table S1 .
8
Quantifying DNMT1 Expression via qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated using the miRNeasy Kit (QIAGEN) and reverse transcription for cDNA was performed using the SuperScript® III First-Strand Synthesis System (18080-051, Invitrogen). TaqMan qPCR (4331182, Applied Biosystems) was performed to measure DNMT1 expression, and SYBR-Green qPCR (4309155, Applied Biosystems, Foster City, CA) was used for other genes. The primers are listed in Supplemental Table . The 18S levels were used for normalization and target expression was analyzed using the ΔCT approach.
9
RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A miRNeasy kit (Qiagen, Frederick, USA) was used for extracting total RNA from 1×106 cells based on provided directions. cDNA was then synthesized from 1 µg of this RNA via M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). A 7900HT real-time PCR system (Applied Biosystems, Foster City, USA) was employed for qPCR analyses, with SYBR Green used to setup triplicate reactions assessing relative mRNA expression. For miRNA expression, TaqMan qPCR was conducted according to provided directions (Applied Biosystems). The 2-ΔΔCt method was used to calculate miRNA and mRNA relative expression, which was normalized to endogenous levels of U6 and GAPDH, respectively.
10
Real-Time PCR Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-Time PCR was performed using the StepOne™ Real-Time PCR system (Applied Biosystems). Primers were designed using Universal Probe library from Roche Diagnostics. Data were analyzed using StepOne™ v2.0 software (Applied Biosystems) and normalized for 18S ribosomal RNA levels. 18S, Ptk2, Yap, Tead1, Tead2, Tead3, Tead4, c-fos, Cdkn2a, Itga5, Itgb5, Itga7, Ccnd1, Cdk4, Timp1, Ctgf, Cyr61, Pax7, MyoD, and Myog gene expression was measured using SYBR® green (Thermo-Fisher Scientific). Primer sequences are provided in Table 1 . IL-6 gene expression was measured using Taqman® qPCR and inventoried Taqman® gene expression assays (Applied Biosystems).
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!