Total RNA was isolated from gastrocnemius using a RNAprep pure animal tissue kit (Tiangen Biotech, Beijing). The cDNA was synthesized using 3 mg of RNA with a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Ishiyama, Japan) following the manufacturer's protocol. The assay was carried out on a CFX96 Real-Time PCR system (Bio-Rad Laboratories) with iTaqTM Universal SYBR Green Supermix (Bio-Rad) and gene-specific primers for IGF-1, mechanistic tar-get of rapamycin (mTOR), myogenic differentiation (MyoD), myoglobin (MyoG), MuRF, and atrogin-1, or GAPDH (Table 2). The PCR conditions were as follows: 95°C for 5 s followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. The quality of RNA was assessed by OD 260 /OD 280 , RNA used in the experiment had an OD 260 /OD 280 ratio between 1.8 and 2.0. Relative expression was first quantified using a standard curve and calculated using the 2 -ΔΔCt , data were normalized to GAPDH mRNA. The primer efficiency was determined based on cycle threshold (Ct) value and melting curve. The Ct value of each mRNA was kept ≤30, and melting curve was controlled single peak. The mRNA and protein expressions were represented by relative expression; the formula used for relative expression was as follows:
Rnaprep pure animal tissue kit
Manufactured by Tiangen Biotech
The RNAprep pure animal tissue kit is a laboratory product designed for the extraction and purification of RNA from animal tissue samples. It is a tool used in molecular biology research and applications.
Automatically generated - may contain errors
2 protocols using rnaprep pure animal tissue kit
1
Quantifying mRNA Expression in Muscle Tissue
2
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA in gastrocnemius was extracted using a RNAprep pure animal tissue kit (Tiangen Biotech, Beijing). cDNA was synthesized using 3 mg of RNA with a PrimeScriptt II 1st Strand cDNA Synthesis Kit (Takara, Ishiyama, Japan) according to the manufacturer's instructions. The assay was carried out on an CFX96 Real-Time PCR system (Bio-Rad Laboratories, USA) with iTaq TM Universal SYBR Green Supermix (BIO-RAD, CA, USA). The qRT-PCR conditions were as follows: 95 °C for 5 s, followed by 40 cycles of 95°C for 10 s, and 60 °C for 30 s. Relative expression was first quantified using a standard curve and 2 -∆∆CT formula, and normalised to GAPDH.
The primers used in this study were shown in Table 2.
The primers used in this study were shown in Table 2.
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!