Total RNA from 2 × 106 MCF-7 cells or exosomal RNA, from media that was collected from 20–24 × 106 cells, were isolated using RNeasy® Mini kit (74104, Qiagen, Hilden, Germany) according to manufacturer’s instructions. OD260/280 nm absorbance ratios were between 1.7 to 2.1 for all RNA isolations. Isolated RNA was treated with DNase-1 (AMPD-1, Sigma, St. Louis, MO, USA) and converted to cDNA by use of iScript™ cDNA synthesis kit (1708891, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Gene amplification was achieved by iTag universal SYBER Green Supermix (172-5122, Bio-Rad, Hercules, CA, USA). Briefly, 10 µL iTaq universal SYBER green Supermix was mixed with 1 µL of cDNA (500 ng/µL), 2 µL of forward and reverse primers (Table 1 ) and 7 µL of nuclease-free water. Three replicates were analysed for each sample. The reaction started at 95 °C for 2 min and followed by 39 cycles of 95 °C for 10 s and 60 °C for 30 s in Thermal Cycler (Bio-Rad, Hercules, CA, USA). Cq data were displayed/analysed using the qPCR software.
Itag universal syber green supermix
Manufactured by Bio-Rad
Sourced in United States
ITag Universal SYBER ® Green supermix is a ready-to-use solution for real-time PCR amplification and detection. It contains all the necessary components, including DNA polymerase, SYBR® Green I dye, and buffer, to perform quantitative PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Abiprism 7500 sequence detection system and analysis software, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis, by Bio-Rad (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
4 protocols using itag universal syber green supermix
1
Quantitative Real-Time PCR Analysis of MCF-7 Cells
2
Quantitative Transcriptome Analysis of Murine Kidney
3
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The mRNA expression of validated genes was determined by real-time quantitative PCR. Brie y, undifferentiated and the Dex-differentiated CAD cells were cultured for 5 days before RNA extraction. RNA concentration and purity were evaluated using a Nanodrop TM 2000 spectrophotometer (Thermo Fisher Scienti c, USA). The cDNA was synthesized using iScript TM cDNA synthesis (catalog number 1708841, Bio-Rad, USA) according to the manufacturer's protocol. Quantitative real-time PCR of the validated genes was performed on an equal amount of cDNA from each condition using iTag Universal SYBER ® Green supermix (catalog number 1725121, Bio-Rad, USA) with the ABIPRISM-7500 sequence detection system and analysis software (Applied Biosystems, USA). The experiments were repeated in triplicate. The mRNA level of GAPDH was used as an internal control. Ct values of the sample were calculated, and transcript levels were analyzed by the 2 -∆∆Ct method. Primers used in this study are listed in Supplementary Table 1.
4
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mRNA expression of validated genes was determined by real-time quantitative PCR. Brie y, undifferentiated and the Dex-differentiated CAD cells were cultured for 5 days before RNA extraction. RNA concentration and purity were evaluated using a Nanodrop TM 2000 spectrophotometer (Thermo Fisher Scienti c, USA). The cDNA was synthesized using iScript TM cDNA synthesis (catalog number 1708841, Bio-Rad, USA) according to the manufacturer's protocol. Quantitative real-time PCR of the validated genes was performed on an equal amount of cDNA from each condition using iTag Universal SYBER ® Green supermix (catalog number 1725121, Bio-Rad, USA) with the ABIPRISM-7500 sequence detection system and analysis software (Applied Biosystems, USA). The experiments were repeated in triplicate. The mRNA level of GAPDH was used as an internal control. Ct values of the sample were calculated, and transcript levels were analyzed by the 2 -∆∆Ct method. Primers used in this study are listed in Supplementary Table 1.
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