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pSilencer 4.1-CMV: Versatile Vectors for Expression of siRNA, miRNA, and mRNA

PDH mRNA (CMV-Gap) or a scrambled sequence (CMV-Scr). (A) Immunostaining of HeLa cells two days after transfection showing reduction of GAPDH levels in cells transfected with CMV-Gap. Green: GAPDH protein. Blue: DAPI stained nuclei. (B) Relative levels of GAPDH expression compared with HeLa cells not transfected (NT).

Figure 2. Expression of miRNA Using pSilencer 4.1-CMV hygro. pSilencer 4.1-CMV was engineered to express mir-16 or mir-124 pri-miRNA. (A) Schematic representation of the mir-16 and mir-124 transcripts. Each transcript included the predicted hairpin at its center and flanking genomic sequences of various sizes. (B) Two days after transfection into HeLa cells, total RNA was isolated with the mirVana miRNA Isolation Kit and 1 g of total RNA was analyzed on a denaturing agarose gel. A Northern blot was used to detect let-7 miRNA, 5.8S and 5S rRNA while miR-16 and miR-124 miRNA were detected by solution hybridization with the mirVana miRNA Detection Kit. All probes were labeled and purified with the mirVana Probe & Marker Kit. NT = not transfected.


Two days after transfection, total RNA was isolated from the cell cultures using the mirVana miRNA Isolation Kit, which provides quantitative yield of small RNAs, including miRNA, siRNA, tRNA, and 5S/5.8S rRNAs. Analysis of miRNA expression by solution hybridization assay with the mirVana miRNA Detection Kit showed a significant overexpression of miR-16 in cells transfected with the pSilencer 4.1-CMV miR-16 vector (Figure 2B). In
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