Michelle A. Anderson, Dean E. Brenner, James M. Scheiman, Diane M

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Reliable Gene Expression Measurements from Fine Needle Aspirates of Pancreatic Tumors Michelle A. Anderson, Dean E. Brenner, James M. Scheiman, Diane M. Simeone, Nalina Singh, Matthew J. Sikora, Lili Zhao, Amy N. Mertens, James M. Rae The Journal of Molecular Diagnostics Volume 12, Issue 5, Pages 566-575 (September 2010) DOI: 10.2353/jmoldx.2010.090107 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Capillary electrophoresis (Agilent Bioanalyzer) of extracted RNA. RNA extracted from pancreatic cancer cell culture (Panc 10.05; lane 2) and fine needle aspirates of patients with pancreatic cancer (lanes 3 to 6). Lane 1 is an RNA ladder. RNA from fine needle aspirates are extensively degraded compared with RNA from pancreatic cancer cell line cultures. The Journal of Molecular Diagnostics 2010 12, 566-575DOI: (10.2353/jmoldx.2010.090107) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Capillary electrophoresis (Agilent Bioanalyzer; A) and RNA yield (B) per pass taken from a nude mouse pancreatic cancer tumor xenograft using variable techniques. A: Lane 1 is RNA extracted from pancreatic cancer cell culture. Lanes 2 to 6 are RNA extracted from 22-gauge endoscopic fine needle aspirates with varying negative pressures as follows: lane 2: no syringe or pressure; lanes 2 to 5: negative pressure using different volume syringes withdrawn to full negative capacity (3 cc, 5 cc, and 10 ml as noted); and lane 6: 10 cc syringe with manufacturer supplied locking-mechanism syringe. Lane 7 is RNA extracted with a 1.5-inch 18-gauge needle. Lane 8 is RNA extracted from a small piece of tumor cut from xenograft. B: Total RNA yield per specimen. The Journal of Molecular Diagnostics 2010 12, 566-575DOI: (10.2353/jmoldx.2010.090107) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Capillary electrophoresis (Agilent Bioanalyzer) for FNA samples taken from a nude mouse breast cancer tumor xenograft using the same techniques used in Figure 2. Lane 1 is RNA extracted from breast cancer (MCF-7) cell culture. Lanes 2 to 6 are RNA extracted from 22-gauge endoscopic fine needle aspirates with varying negative pressures as follows: lane 2: no syringe or pressure; lanes 2 to 5: negative pressure using different volume syringes withdrawn to full negative capacity (3 cc, 5 cc, and 10 ml as noted); and lane 6: 10 cc syringe with manufacturer supplied locking-mechanism syringe. Lane 7 is RNA extracted with a 1.5-inch 18-gauge needle. Lane 8 is RNA extracted from a small piece of tumor cut from xenograft. The Journal of Molecular Diagnostics 2010 12, 566-575DOI: (10.2353/jmoldx.2010.090107) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Agilent capillary electrophoresis PCR products FNA samples from three pancreatic tumors. Multiplex PCR for GAPDH targeting varying (100, 200, 300, 400, 500, 600, and 700 bp) lengths of mRNA was performed by using different forms of template. Lanes 1 to 4 are from tumor number 1; lanes 5 to 8 are from tumor number 2; and lanes 9 to 12 are from tumor number 3. Templates are as follows: lanes 1, 5, and 9: untreated RNA; lanes 2, 6, and 10: RNA that was DNase treated; lanes 3, 7, and 11: cDNA made from untreated RNA; and lanes 4, 8, and 12: cDNA made from DNase-treated RNA. Results show that there are small amounts of genomic DNA within the extracted RNA before DNase treatment as shown by the presence of bands of varying lengths in lanes 1, 5, and 9 and the absence of bands, following DNase treatment, in lanes 2 and 10. The persistence of bands in lane 6 shows that the DNase treatment was unsuccessful in tumor number 2. The persistence of the 100 bp band in the cDNA made from RNA that had been DNase treated (lanes 4 and 12) demonstrates that there are fragments of mRNA in these specimens that can be successfully amplified. L = DNA ladder. The Journal of Molecular Diagnostics 2010 12, 566-575DOI: (10.2353/jmoldx.2010.090107) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions