Magnetic mRNA Isolation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- If the lysate viscous than normal. Pass these cell or tissue lysates several times through a 21-gauge needle. If the yield of mRNA is low Increase the ratio of beads to Lysis/Binding Buffer or add RNase-free Proteinase K to the sample lysate directly before the 10 minute binding incubation

Protocol tips
- If the lysate viscous than normal. Pass these cell or tissue lysates several times through a 21-gauge needle. If the yield of mRNA is low Increase the ratio of beads to Lysis/Binding Buffer or add RNase-free Proteinase K to the sample lysate directly before the 10 minute binding incubation

Publication protocol

mRNA of rat olfactory epithelium (OE) was isolated using Dynabeads Magnetic Beads (Invitrogen; Life Technologies). cDNA was made by reverse transcription using random hexamer primers (Thermo Fisher Scientific) and SuperScript III Reverse transcription (Invitrogen). The primers used for full-length cloning (Table S2) spanned the entire open reading frame of rat ANO 1 (Hartzell et al., 2009) and ANO 2 (Stephan et al., 2009). An NheI site was introduced at the 5′ end and a BamHI (ANO 1) or SmaI (ANO 2) site at the 3′ end of the fragments for fusing YFP to the C terminus of ANO 1 and ANO 2 in the expression vector pEYFP-N1 (Takara Bio Inc.). The resulting constructs were verified by sequencing. Rat ANO 1 protein sequence from OE corresponds to database entry NP_001101034 starting with amino acid (aa) position M59. In addition, V528 to C531 is replaced by the aa sequence EAVK (5′-GAAGCTGTCAAG-3′) due to an incorrect splicing site prediction in rat. Rat ANO 2 from OE corresponds to XP_003753992 starting with aa position M23. The photoreceptor isoform of mouse ANO 2 in the expression vector pCMV-Sport6 was obtained from the German Science Centre for Genome Research (RZPD; clone identification: IRAVp968H1167D; see also Pifferi et al., 2009).

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