Supplementary Material
Appendix S2: Experimental procedures
Strand-specific RNase digests
To analyze whether miRNAs are existing as double- or single stranded molecules,
strand-specific RNase digests were performed. RNA from 1200 µl phloem sap was
isolated by using Trizol LS reagent (Invitrogen). The resuspended RNA was divided to
3 reaction tubes with equal amounts of total RNA (20 µg) in a volume of 20 µl 1x
RNase structure buffer (provided with the RNases, Ambion). 8 µl (8 units) of single
strand-specific T1, double strand-specific V1 RNase (both from Ambion), or DEPCtreated water as a control, respectively, were added to the different reaction tubes. After
incubation for 1 hour at room temperature, the digests were stopped by adding 25 µl of
100 % formamide to all samples, and subjected to RNA gel blot analysis. To confirm
the activity of the ds-specific V1 enzyme, similar experiments were performed using
synthetic double stranded miR164a (MWG Biotech) in a total volume of 10 µl.