RNA was extracted from the cells using TRIzol (Invitrogen) following the manufacturer’s protocol. The total RNA was reverse transcribed to cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo scientific, USA). SYBR Green quantitative PCR master mix from Qiagen was used for quantitative RT-PCR analysis on the Applied Biosystems 7500 real-time RT-PCR system from Life Technologies (Grand Island, NY, USA). The expression of microRNAs was measured by utilizing TaqMan microRNA assay kit from Applied Bio-systemsTM (life Technologies, Carlsbad, California, USA). As internal normalization controls for mRNA and miRNA, GAPDH and U6 were used. The relative quantitation of miRNA expression was calculated using the 2−∆∆Ct method, with the ∆∆Ct value determined as the difference between the Ct value of the treatment group and the control group. A list of primers used in this study can be found in Supplemental Material Tables S1 and S2 .
Sybr green quantitative pcr master mix
Manufactured by Qiagen
Sourced in United States
SYBR Green quantitative PCR master mix is a reagent used in quantitative PCR (qPCR) experiments. It contains the necessary components for DNA amplification and detection, including a DNA polymerase, buffer, and SYBR Green dye, which binds to double-stranded DNA and emits fluorescence upon binding. This master mix enables the quantification of target DNA sequences in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 7500 real time rt pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
β actin, by Qiagen (1 mentions)
Cfx96 rt pcr detection system, by Bio-Rad (1 mentions)
Rneasy mini rna isolation kit, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
3 protocols using sybr green quantitative pcr master mix
1
Quantitative Analysis of miRNA Expression
2
Gene Expression Analysis of NFAT-Regulated Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression was quantified using CFX 96 RT-PCR Detection System (BioRad, Hercules, CA). Target sequences were amplified using commercially available RT2 (link) quantitativePCR Primer Sets (Qiagen, Frederick, MD) with RT2 (link) SYBR green quantitativePCR Mastermix (Qiagen) according to the manufacturer’s protocol. For quantitation of messenger RNA expression levels, we used the 2(−∆∆CT) method. Gene expression was normalized to β-actin (Qiagen).
The residual gene expression after tac intake was calculated as T1.5/T0*100, where T0 is the adjusted number of transcripts at tac predose level and T1.5 is the number of transcripts 1.5 hours after drug intake. For all 3 genes, the RE was averaged and presented as mean RE (MRE) of NFAT-regulated genes.
The residual gene expression after tac intake was calculated as T1.5/T0*100, where T0 is the adjusted number of transcripts at tac predose level and T1.5 is the number of transcripts 1.5 hours after drug intake. For all 3 genes, the RE was averaged and presented as mean RE (MRE) of NFAT-regulated genes.
3
Quantifying Cytokine-Induced Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following cell synchronization for 24 h, cells were left unstimulated or were stimulated for 12, 24 or 48 h with 50 ng/mL TNF-α. Total RNA was extracted using RNeasy Mini RNA isolation kits (Qiagen, Valencia, Spain) in accordance with the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using a High-Capacity cDNA Reverse transcription Kit (Applied Biosystems, Waltham, MA, USA) per the manufacturer’s protocol. CCL11, CCR3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were purchased from Qiagen. Quantitative PCR was performed using SYBR Green quantitative PCR master mix (Qiagen) on a C1000 thermal cycler (Bio Rad, Hercules, CA, USA) as described previously5 (link). The efficiency of the primer assays was guaranteed by the manufacturer to be > 90%. Each reaction was measured in triplicate, and the data were normalized to the expression levels of the housekeeping gene GAPDH. The ratio of each mRNA relative to the GAPDH mRNA was calculated using the ΔΔ threshold cycle method.
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!