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. 2013 Apr 29;8(4):e62125. doi: 10.1371/journal.pone.0062125

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© 2013 Ehsani et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A) (Upper panel) Lanes 2 and 4 illustrate detection of EGFP mRNA (upper band) and pre-mRNA (lower band) in RNA samples isolated from anti-U5-200kD shRNA and irrelevant shRNA treated cells, respectively. Lanes 1 and 3 are minus RT controls. (Middle panel) RT-PCR analysis of EGFP mRNA expression. Lanes two and four demonstrate expression of the histone (top band) and spliced EGFP mRNAs in anti-U5-200kD shRNA and irrelevant anti-EGFP rev shRNA treated 293/EcR cells. Lanes 1 and 3 are minus RT reactions. (Lower panel) Semi-quantitative RT-PCR analysis of the EGFP mRNA expression. B) RT-PCR detection of alpha-tubulin pre-mRNA and histone mRNA. (Upper panel) The lower band in Lane 2 is the alpha-tubulin pre-mRNA product from samples treated with U5-200kD specific shRNA and the irrelevant anti-Rev shRNA, respectively. The upper bands are the histone products used for normalization. Lanes 1 and 3 are minus RT controls. The 100bp size marker was used for product size verification. (Middle panel) RT-PCR detection of the alpha-tubulin and histone mRNAs. The lower bands in Lanes 2 and 4 are alpha-tubulin products from samples treated with the U5-200kD specific hairpin and the irrelevant anti-Rev shRNAs, respectively. The upper bands are the histone products used for normalization. Lanes 1 and 3 are minus RT controls. The 100bp size marker was used for product size verification. (Lower panel) Semi-quantitative analysis of alpha-tubulin mRNA expression. Semi-quantitative analysis of alpha-tubulin mRNA expression normalized against histone mRNA expression revealed only a 16% reduction in the expression of the alpha-tubulin mRNA in the splicing defect background.