Differential expression of miRNAs in apple tissues. A, Gel blot analyses of miR156, miR159, miR166, miR167 and miR172 expression. Low molecular weight RNA purified from 20 μg of total RNA was separated by electrophoresis, transferred and hybridised with an appropriate antisense probe. An ethidium bromide-stained prominent band of small RNA was used as the loading control. B, Stem-loop RT-PCR analyses of miR156, miR159, miR166, miR167 and miR172 expression. 10 ng of total RNA was used in reverse transcription and subsequent PCR amplification. Number of PCR cycles is indicated on the right. Note that 28 cycles of PCR corresponds approximately to gel-blot analysis results. MdACT mRNA was amplified using standard RT-PCR. C, Stem-loop RT-PCR analyses using 10 ng total RNA of miR160, miR162, miR164, miR168, miR169, miR171, miR390, miR393, miR394, miR396, miR397, miR398, miR403, miR408, miR475, and miR476 expression. Number of PCR cycles is indicated on the right. MdACT mRNA was amplified using standard RT-PCR. SA, shoot apex; L, leaf; S, stem; P, periderm; Ph, phloem; X, xylem (the last three layers were collected by peeling from the stem); sRNA, small RNA loading control; MdACT, apple ACTIN amplification product.