The spheroids embedded in the collagen matrix were lysed using Trizol Reagent (Life Technologies). Then, the RNA was isolated from samples using RNeasy Micro kits (Qiagen) according to the manufactural protocols. Subsequent RNA concentration was then measured with a NanoDrop (Thermo Scientific). Reverse transcription to cDNA was performed using Tetro cDNA Synthesis Kit (Bioline). Real-time quantitative PCR was performed with FastStart universal SYBR Green Master reagents (Rox) (Roche) in a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). Fold changes of transcripts in D5-cultured MCF7 spheroids embedding in collagen matrix relative to D0-cultured MCF7 free spheroids were determined by ΔΔCt to study the gene expression of growth, cell cycle arrest, apoptosis, and EMT. The primers sequences used in this study are listed in Table 1 .
Faststart universal sybr green master reagents rox
Manufactured by Roche
Sourced in Germany
The FastStart Universal SYBR Green Master reagents (Rox) are a ready-to-use solution for real-time PCR amplification and detection. The reagents contain SYBR Green I dye, FastStart Taq DNA Polymerase, and necessary PCR components, including ROX reference dye.
Automatically generated - may contain errors
Lab products found in correlation
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Tetro cdna synthesis kit, by Meridian Bioscience (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
2 protocols using faststart universal sybr green master reagents rox
1
Gene Expression Analysis of 3D Cultured MCF7 Spheroids
2
Quantitative PCR Analysis of Gene Expression
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Total RNA
was purified from samples using an RNeasy mini kit (Qiagen, Singapore).
tRNA was then transcribed into cDNA using a Tetro cDNA synthesis kit
(Bioline, U.K.). Real-time quantitative PCR was performed with FastStart
Universal SYBR Green Master reagents (Rox) (Roche, Germany) in a ViiA
7 Real-Time PCR System (Thermo Fisher Scientific).
Fold changes
of transcripts in U937 cells relative to untreated control samples
were determined by ΔΔCt while log2(GAPDH-gene) was used
for hepatocyte gene expression. The primers used for human hepatocytes
and U937 cells are listed inTables S4 and S5 , respectively.
was purified from samples using an RNeasy mini kit (Qiagen, Singapore).
tRNA was then transcribed into cDNA using a Tetro cDNA synthesis kit
(Bioline, U.K.). Real-time quantitative PCR was performed with FastStart
Universal SYBR Green Master reagents (Rox) (Roche, Germany) in a ViiA
7 Real-Time PCR System (Thermo Fisher Scientific).
Fold changes
of transcripts in U937 cells relative to untreated control samples
were determined by ΔΔCt while log2(GAPDH-gene) was used
for hepatocyte gene expression. The primers used for human hepatocytes
and U937 cells are listed in
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