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. 2011 Dec 19;109(1):279–284. doi: 10.1073/pnas.1114204109

Fig. 4.

Fig. 4.

m169 is a 1,669-nt transcript with expression kinetics that correlate with the degradation of miR-27. (A) Schematic of m169 gene locus based on 5′ and 3′ RACE. (B) Left, validation of m169 transcriptional start site based on primer extension using a 20-nt primer starting at position 229,039. (B) Center, PCR product of the m169 transcript using a forward primer at the transcriptional start site and reverse primer at the 3′ end; the cDNA was created with an oligo(dT) primer. (B) Right, Northern blot analysis with a 80-nt probe that spans the miR-27-binding site in m169. RNA was collected from NIH 3T3 cells mock-infected (−) or infected with MCMV at MOI = 5 (+) for 48 h and then resolved on a formaldehyde-agarose gel. (C) Left, Northern blot analysis with RNA collected from NIH 3T3 cells mock infected (−) or infected (+) with MCMV (MOI = 5) for the indicated times. (C) Right, Northern blot analysis with RNA collected from NIH 3T3 cells mock-infected or infected with MCMV (MOI = 5) for 16 h in the absence and presence of 5 μg/mL actinomycin D (ACT); 5 μg of total RNA was loaded per lane. (D) Absolute quantification of m169 and miR-27 at the indicated time points postinfection (MOI = 5) based on qRT-PCR, using synthetic miR-27 and in vitro transcribed m169 as standards.