1. Analytical Validation of Androgen Receptor Splice Variant 7 Detection in a Clinical Laboratory Improvement Amendments (CLIA) Laboratory Setting 
Parvez M. Lokhandwala, Stacy L. Riel, Lisa Haley, Changxue Lu, Yan Chen, John Silberstein, Yezi Zhu, Gang Zheng, Ming-Tseh Lin, Christopher D. Gocke, Alan W. Partin, Emmanuel S. Antonarakis, Jun Luo, James R. Eshleman 
The Journal of Molecular Diagnostics 
Volume 19, Issue 1, Pages (January 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Androgen receptor splice variant 7 (AR-V7) test workflow. Patient blood samples in a minimum of two (preferably three) yellow-top tubes are obtained. Two tubes are used to test the patient in duplicate using our assay as follows. Circulating tumor cells (CTCs) are enriched from the blood samples and mRNA is isolated. Green lines and red lines designate AR-FL and AR-V7 mRNA, respectively. The RNA is reverse-transcribed to cDNA. cDNA is used in a multiplexed PCR reaction followed by electrophoresis on TapeStation to detect the presence of CTCs. cDNA also is used as a template for real-time quantitative PCR to amplify six target regions to investigate the presence of CTCs and AR-V7. Any amplified prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), AR-FL, or AR-V7 products were confirmed by Sanger sequencing. Purple horizontal bar shows 1500-bp size standard; green horizontal bar, 25-bp size standard. ACTB, beta-actin; EGFR, epidermal growth factor receptor.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
A: Microsatellite profile of unspiked donor blood DNA. B: Microsatellite profile of LNCaP95 DNA. C: Microsatellite profile of donor blood spiked with 10,000 LNCaP95 cells after enrichment. D: Microsatellite profile of donor blood spiked with 50,000 LNCaP95 cells after enrichment. After CTC-enrichment the majority of the DNA (≥90%) is derived from the prostate cancer cells, showing effective enrichment.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Different estimated numbers of LNCaP95 cells in duplicate were spiked into a healthy donor blood sample and detected using the Adna ProstateCancerDetect kit, as well as our assay. A: TapeStation image after ProstateCancerDetect kit showing amplification of prostate-associated antigens [prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), beta-actin (ACTB)] from samples spiked with 5 or more cells. B: The average cycle threshold (Ct) value of androgen receptor splice variant 7 (AR-V7) real-time quantitative PCR amplification correlates inversely with the number of spiked LNCaP95 cells. Purple horizontal bar shows 1500-bp size standard; green horizontal bar, 25-bp size standard. Experiments were performed in duplicate and error bars are ± 1 SD. Avg, average.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Real-time quantitative PCR amplification using diluting concentrations of input LNCaP95 RNA was performed. The graph of average cycle threshold (Ct) value versus log10 (input RNA concentration) shows amplification of both AR-FL and AR-V7 from up to 1 pg (0 on log10 scale) of input LNCaP95 RNA. Experiments were performed in duplicate and error bars are ± 1 SD. AR, androgen receptor; Avg, average; FL, full length; V7, splice variant 7.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions