Fig. 4.

Detection of mature mir-20a, chr19 RNA and Malat-1. (a) Visualization of mature mir-20a and a larger RNA in 12% polyacrylamide gel. In lactating (LMG) or virgin mammary glands (MG) from wild type (Wt) mice, the levels of mature mir-20a at about 65 bp are lower than the levels in mammary tumors (MT) and proliferating mammary glands (MMTV) from MMTV-c-myc transgenic mice. An additional band occurring at about 100 bp shows a decreased level in MMTV-c-myc mammary glands with decreases to undetectable levels in the tumors. (b) PCR amplification of hexmer primer-derived RT products with primers specific for Malat-1 does not show any difference between the tumors and normal mammary tissues. (c) Cloning the 65-bp band to TOPO vector followed by plasmid sequencing confirms that this band is mature mir-20a (small-case letters at the 5′-end of the sequence). (d) Plasmid sequencing also shows that the 100-bp band is generated from mis-priming of the mir-20a sequence (used as the forward primer) with part of Malat-1 (the 36-bp sequence in small-case letters), which is a large non-coding RNA transcribed from chr19