Detection of miRNAs in tissue and phloem sap. A. Gel blot analyses of miRNA expression in pumpkin shoot apex (SA), leaf (L) and stem (S). An ethidium bromide-stained prominent band of tRNA was used as the loading control (LC). B. Stem-loop end-point RT-PCR analyses of miRNA expression. miRNAs were amplified using 28 cycles of PCR. Pumpkin RUBISCO (CmRBS) mRNA was amplified using 30 cycles of standard PCR. C. Stem-loop end-point RT-PCR analyses of miRNA expression in pumpkin phloem sap. The number of PCR cycles is indicated on the top. miR156, miR159 and miR167, but not miR171 were detected.