RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- 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
- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
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

Total RNA isolation and cDNA preparation from RMA-S/B7-1.Trh4 and RMA-S Trh4/pUB cells were performed using an RNeasy Mini Kit (Qiagen, MD, USA). Five hundred nanograms of purified total RNA were used to synthesize cDNA using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, USA). Quantitative PCR on short and long transcripts of Trh4 was done as described previously [13]. SensiMix SYBR No-ROX kit from GC Biotech Bioline (Alphen aan den Rijn, NL) was used in a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) and results were analyzed using Bio-Rad CFX manager software. Long Trh4 (Lass5) transcripts were amplified with Power SYBR Green Master Mix (Applied Biosystems) on a GeneAmp 7300 System (Applied Biosystems).

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Manufacturer protocol

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

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