Toward Rare Blood Cell Preservation for RNA Sequencing

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Presentation transcript:

Toward Rare Blood Cell Preservation for RNA Sequencing Sanja Vickovic, Afshin Ahmadian, Rolf Lewensohn, Joakim Lundeberg The Journal of Molecular Diagnostics Volume 17, Issue 4, Pages 352-359 (July 2015) DOI: 10.1016/j.jmoldx.2015.03.009 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Protocol workflow. Cell-line samples were either spiked into whole blood or processed separately. Fixation was performed. After fixation, either immunomagnetic separation or prolonged storage was tested. All RNA material was checked for RNA integrity in a quality control step. Stranded RNA TruSeq libraries were prepared, sequenced, and the data analyzed. Downstream data analysis included gene expression and fusion transcript analysis. The Journal of Molecular Diagnostics 2015 17, 352-359DOI: (10.1016/j.jmoldx.2015.03.009) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Quality control of the integrity of RNA molecules (RIN) values for the A-549 and MCF-7 cancer cell lines. A: The graph depicts the effects of fixation and storage of of A-549 fresh and A-549 fixed at −20°C for different time periods. B: The graph depicts the effects of fixation (MCF-7 fresh and MCF-7 fixed) and epitope availability after fixation (IMS fresh and IMS fixed). FU, fluorescence units; IMS, immunomagnetic separation. The Journal of Molecular Diagnostics 2015 17, 352-359DOI: (10.1016/j.jmoldx.2015.03.009) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Comparison of quality control capillary electropherograms from 2% PFA and 10% NBF after 14 (A) and 21 days (B) of storage. FU, fluorescence units; NBF, neutral buffered formalin; nt, nucleotides; PFA, paraformaldehyde. The Journal of Molecular Diagnostics 2015 17, 352-359DOI: (10.1016/j.jmoldx.2015.03.009) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Gene body coverage plot for the MCF-7 cancer cell line. Pearson's correlation (r2 = 0.9918) indicated between the fresh and fixed sample pair. The Journal of Molecular Diagnostics 2015 17, 352-359DOI: (10.1016/j.jmoldx.2015.03.009) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Gene expression plots with a linear fitted line. Pearson's correlation for MCF-7 cancer cell line pair (A), IMS cancer cell line pair (B), spiked blood pair (C), and A-549 cancer cell line pair (D). P < 2.2 × 10–16 in all of the sample pairs. The Journal of Molecular Diagnostics 2015 17, 352-359DOI: (10.1016/j.jmoldx.2015.03.009) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Relative gene expression of a selected panel of genes in spiked and unspiked blood samples. The expression measurements are based on RT-qPCR studies and demonstrate that the relative expression of CDH1, CD24, ERBB2, and KRT19 in spiked blood samples is much higher than in unspiked blood. The Journal of Molecular Diagnostics 2015 17, 352-359DOI: (10.1016/j.jmoldx.2015.03.009) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Circos plot for the spiked blood sample. A: Spiked blood fresh sample. B: Spiked blood fixed sample. Grey lines indicate intrachromosomal fusions. n indicates the number of fusions. The Journal of Molecular Diagnostics 2015 17, 352-359DOI: (10.1016/j.jmoldx.2015.03.009) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions