The single clone of MG1363, pGS/MG1363 and pGSMT/MG1363 was inoculated respectively in tube containing 5 mL of GM17 broth medium at 30 °C for 1 d. Cells of 1 mL were collected by centrifugation at 12,000 × g for 1 min at 4 °C. The pellets were lysed and the total RNA was extracted using HiPure Bacterial RNA Kit (Magen, Guangzhou, P.R. China). Total RNA of 1 μg was used as the template for cDNA synthesis primed with random hexamers (Bio-Rad, Hercules, CA, USA). The reaction mixture was incubated at 42 °C for 30 min followed by 5 min at 85 °C. The PCR conditions were as follows: one cycle at 94 °C for 3 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with an additional extension at 72 °C for 7 min. Three DNA fragments (mt, gst-sumo and 16s rRNA) were performed for each sample using different primers (Supplementary Table S1 ) and the PCR products were checked on 2% (w/t) agarose gel electrophoresis.
Random hexamers
Manufactured by Bio-Rad
Sourced in United States
Random hexamers are short DNA sequences consisting of six randomly ordered nucleotides. They are commonly used in molecular biology applications as primers for reverse transcription and DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using random hexamers
1
RNA Extraction and cDNA Synthesis
2
Quantifying mRNA Levels in Mice Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For gene expression analyses, mRNA was obtained from the hypothalamus tissue of adult mice and GT1-7 cells using RNeasy Plus Mini Kits (Qiagen, Chatsworth, CA, US). We used 1000 ng total mRNA as the template for cDNA synthesis primed with random hexamers (Bio-Rad, Richmond, CA, US). Then, the reaction mixture was diluted at 1:2, and 2 μl of the diluted template was used per 20 μl of the real-time quantitative PCR assay using the Bio-Rad SsoAdvanced™ Universal SYBR (Bio-Rad 172–5274, Mississauga, ON, Canada). The Bio-Rad CFX Connect Real-Time system and the Bio-Rad CFX Manager Software (version 2.0) were used to collect the PCR data. Results are presented as linearized values normalized against β-actin. The primers (Table S2) were synthesized by the Beijing Genomic Institute (BGI).
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!