RNeasy Plus Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Publication protocol

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).



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Manufacturer protocol

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