Figure 1. Construction and expression of miR-328.

(A) Diagram of a construct expressing GFP, a neomycin-resistance gene, and two hairpin structures of pre-miR-328. The bolded and capitalized letters represent two restriction sites BglII and HindIII. The bolded lower case sequence represents an artifact sequence inserted between two pre-miRNAs. Six “t”s were added to terminate transcription. A few lower case letters represent changes of nucleotides that are not part of the mature miR-328 but are useful for PCR purpose. (B) RT-PCR of pre-miR-328 using RNA prepared from A431 cells stably transfected with miR-328 and a control vector, using two primers miR-328N and miR-328C, confirming expression of pre-miR-328. As a control, Gapdh was amplified with two primers huGapdh131F and huGapdh380R. RT-PCR of mature miR-328 using RNA prepared from A431 cells stably transfected with miR-328 and a control vector, confirming proper processing of miR-328. (C) Real-time PCR analysis confirmed expression of mature miR-328. **, p<0.01. Error bars, SD (n = 3). The end products of real-time PCR were subjected to agarose gel electrophoresis (lower).