RNeasy Plus Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- On-column digestion is preferable - For high gDNA elimination spin at 6000rpm for 5 min instead of the quick high speed spin	- 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.

Protocol tips
- On-column digestion is preferable - For high gDNA elimination spin at 6000rpm for 5 min instead of the quick high speed spin
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

2.2. RNA extraction
C6, PC12 and CGC cultures were washed with 1× Phosphate-Buffered Saline (PBS) prior to RNA isolation. The cells were lysed directly on the culture dish using the lysis buffer provided in Qiagen’s RNeasy Mini Plus kit for total RNA isolation, and genomic DNA was removed using Qiagen’s gDNA Eliminator columns following the manufacturer’s protocols (Qiagen, Toronto, ON, Canada). Total RNA from juvenile rat hippocampus was extracted using TRIzol (Invitrogen, Burlington, ON, Canada), and further purified using Qiagen’s RNeasy Mini Plus kit and gDNA Eliminator columns following the manufacturer’s protocols. Total RNA samples from various juvenile and adult tissues and different rat strains (n = 34) were purchased from Zyagen (San Diego, CA, USA), see Table S2.

2.3. Evaluation of RNA purity and integrity
A Nanodrop 1000 spectrometer (Thermo Fisher Scientific, Ottawa, ON, Canada) was used to measure absorbance at 260 nm (A260) to evaluate RNA concentration. RNA purity was estimated using the A260/A280 ratio and only samples presenting a ratio greater than 1.8 were kept for further analyses. RNA integrity was assessed by an Agilent 2100 Bioanalyzer, using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Mississauga, ON, Canada). The Solaris RNA Spike Control kit (Thermo Fisher Scientific, Cat# K-002200-C1-100) was used to assess the presence of inhibitors in a subset of RNA samples (Fig. S2). A PCR-based approach developed in-house [16] was used to assess gDNA contamination in RNA samples purchased from a commercial supplier. All the samples tested proved to be free from inhibitors and gDNA contamination.

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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Plus Mini Kit below.

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