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

Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and RNeasy Plus Mini Kit (Qiagen). Genomic DNA was eliminated with RNase free DNase 1 (Roche), and total RNA was then subjected to another round of purification by phenol solution with pH4.3 ± 0.2 (Sigma-Aldrich). For pluripotent gene expression analysis, cDNA was generated by SuperScript II reverse transcriptase (Invitrogen) with random hexamers (Invitrogen) from 1 µg of total RNA, and real-time PCR was performed with GoTaq qPCR Master Mix (Promega).

For Gdf3 RNA was isolated from whole bone marrow mononuclear cells or CD45.2+ / LSK cells using TRIzol reagent (Invitrogen) and RNeasy mini kit (QIAGEN). cDNA was synthesized with Superscript First-Strand Synthesis System according to manufacturer's instructions (Invitrogen). qPCR was performed with SYBR reagent (Bio-Rad).

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Manufacturer protocol

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