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Figure 2. Poly-A tail analysis of selected mRNAs.mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2. Image published in: Blower MD et al. (2013) Image reproduced on Xenbase with permission of the publisher and the copyright holder. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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