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. 2017 Nov 20;7:15833. doi: 10.1038/s41598-017-16177-9

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Construction and expression of vectors containing human genomic β-globin. (a) Schematic diagram of expression vector containing wild-type human genomic β-globin (gBglo-WT) and plasmids derived from gBglo-WT by introducing premature termination codons (PTCs) at the indicated positions. (b) Schematic of location of semi-qPCR amplicon. Semi-qPCR analysis of precursor β-globin mRNA, with EGFP serving as internal control (c) qPCR analysis of mRNA isolated from HeLa cells transfected with the β-globin constructs. (d) RNA-FISH-mediated evaluation of the cytoplasmic localization of β-globin mRNAs from gBglo-WT, gBglo-P39, and gBglo-P66. (e) Immunofluorescence intensity of β-globin was quantified and normalized that of GAPDH mRNA. Every experiment contributing to Fig. 1 was performed three independent times and the results of one representative experiment are shown. One-way ANOVA with a post-hoc test was performed to compare multiple means. ***p < 0.001. Error bars in (c) and (e) represent the SD of the mean of three independently performed qPCR analyses and the SD of the mean of normalized β-globin intensities from 10 randomly selected cells, respectively.