Reverse-transfected cells were collected at 48 h and processed using Fast SYBR Green Cells-to-Ct Kit (Ambion) per manufacturer’s instructions. QuantiTect Primer Assays (Qiagen) were used (Supplementary Table 3
Fast sybr green cells to ct kit
Manufactured by Thermo Fisher Scientific
The Fast SYBR Green Cells-to-Ct Kit is a rapid and sensitive kit designed for the direct quantification of gene expression from cultured cells. The kit utilizes SYBR Green I dye for the detection and quantification of DNA during real-time PCR.
Automatically generated - may contain errors
2 protocols using fast sybr green cells to ct kit
1
Quantitative PCR Assay for Gene Expression
2
Osteogenic Differentiation of MC3T3-E1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the purpose of the real-time polymerase chain reaction (qPCR)
analysis, MC3T3-E1 cells were first seeded on PCL/HAp films in 96-well
plates at the density of 3 · 104 cells per well and
cultured in 100 μL of the above-mentioned AA-free medium for
48 h. The AA-free medium was then substituted with the AA-supplemented
medium. After an additional 48 h incubation period, cell lysis, reverse
transcription (Bio-Rad) and qPCR (Applied Biosystems, StepONEPlus)
were performed using the Fast SYBR Green Cells-to-CT kit (Ambion)
in accordance with the manufacturer’s instructions. Each experiment
was done in triplicates and each experimental replica was analyzed
for mRNA expression in triplicates too (n = 3 ×
3). The expressions of one housekeeping gene, β-actin (ACTB), and six additional ones, including osteocalcin (BGLAP), osteopontin (BSP-1), procollagen
type I (Col I), alkaline phosphatase (ALP), and the transcription factors, Runx2 and TGFβ-1 were analyzed. Table1 shows the primer pair sequences used. The real-time PCR results
were analyzed using the ΔΔCt method and all
the data were normalized to ACTB expression levels.
analysis, MC3T3-E1 cells were first seeded on PCL/HAp films in 96-well
plates at the density of 3 · 104 cells per well and
cultured in 100 μL of the above-mentioned AA-free medium for
48 h. The AA-free medium was then substituted with the AA-supplemented
medium. After an additional 48 h incubation period, cell lysis, reverse
transcription (Bio-Rad) and qPCR (Applied Biosystems, StepONEPlus)
were performed using the Fast SYBR Green Cells-to-CT kit (Ambion)
in accordance with the manufacturer’s instructions. Each experiment
was done in triplicates and each experimental replica was analyzed
for mRNA expression in triplicates too (n = 3 ×
3). The expressions of one housekeeping gene, β-actin (ACTB), and six additional ones, including osteocalcin (BGLAP), osteopontin (BSP-1), procollagen
type I (Col I), alkaline phosphatase (ALP), and the transcription factors, Runx2 and TGFβ-1 were analyzed. Table
were analyzed using the ΔΔCt method and all
the data were normalized to ACTB expression levels.
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!