Evidence for Antisense Transcription Associated with microRNAs in Arabidopsis

Exploring miRNA-related antisense transcription in Arabidopsis through RNA transcript profiling of smRNA pathway-defective mutants on a custom high-resolution oligonucleotide array. Two custom arrays were designed with 15,000 oligonucleotide (25-mer and 36-mer) probes in each array. The probes were selected tiling 200 base upstream and downstream (3 n.t. resolution) of miRNA target sites in Arabidopsis thaliana. The arrays were fabricated by Agilent Technologies, Santa Clara, CA. The 22 target genes on the arrays were chosen based on the abundance of associated smRNAs that mapped to the loci, the amplitude of the antisense transcription signals in published whole genome tiling microarray experiments and a representative cross section of miRNA families. For 15k arrays with 24 selected miRNA targets, a dye swap loop experiment design was utilized with 12 blocks for 7 genotypes on two chip arrays. The details of the experimental design are in Supplemental Table V of the manuscript. For the hen1-1 versus Ler-0 experiment, a dye swap with two versus three biological replicates and four array blocks was performed.