Tissue Banking of Diagnostic Lung Cancer Biopsies for Extraction of High Quality RNA Malcolm H. Lawson, MBBS, MRCP, Doris M. Rassl, MBBS, FRCPath, Natalie.

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Tissue Banking of Diagnostic Lung Cancer Biopsies for Extraction of High Quality RNA Malcolm H. Lawson, MBBS, MRCP, Doris M. Rassl, MBBS, FRCPath, Natalie M. Cummings, MBChB, MRCP, Roslin Russell, PhD, Jaymin B. Morjaria, MD, MRCP, James D. Brenton, PhD, FRCP, Gillian Murphy, PhD, FMedSci, Robert C. Rintoul, PhD, FRCP Journal of Thoracic Oncology Volume 5, Issue 7, Pages 956-963 (July 2010) DOI: 10.1097/JTO.0b013e3181ddbbe9 Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Flow chart showing tissue banking process. Journal of Thoracic Oncology 2010 5, 956-963DOI: (10.1097/JTO.0b013e3181ddbbe9) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Optimization of RNA extraction method. To establish the best method for sample collection and storage, paired biopsies were taken from the same tumor in a series of patients. Panel A, RNA yield in the presence or absence of a DNA elimination step. Yield for each biopsy for three pairs is plotted. DNA elimination reduced the apparent yield by 80%. Yield of RNA from biopsies taken with endobronchial biopsy forceps (FOB, Panel B) and by TBNA (Panel C); the dotted line represents an RNA yield of 500 ng. Panel D, Fold change in yield with RNAlater treatment for biopsies taken by TBNA or FOB. Bars represent median and interquartile range; dotted line represents a fold change of 1 (equivalence). Yield was increased by storage in RNAlater for both FOB (p = 0.03) and TBNA (p = 0.04) samples. Panels E and F show the RIN of RNA extracted from FOB and TBNA biopsies, respectively. The dotted line represents a RIN of 6. The RIN was higher in 11 of the 16 biopsy pairs tested (p = 0.01). FOB, fiberoptic bronchoscope; TBNA, transbronchial needle aspiration; RIN, RNA integrity number. Journal of Thoracic Oncology 2010 5, 956-963DOI: (10.1097/JTO.0b013e3181ddbbe9) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Assessment of RNA degradation because of different biopsy techniques. Twelve to 16 biopsies were taken from each of three surgically resected lung cancer specimens. Half were taken using endobronchial biopsy forceps, and half were dissected from the tumor using a scalpel. All biopsies were of approximately the same size. For each type of biopsy, half were stored snap frozen and half in RNAlater. RNA was subsequently extracted as per protocol to compare yield (Panel A) and quality (Panel B). Bars represent mean ± 95% confidence interval. Dotted lines represent a yield of 500 ng (Panel A) and RIN = 6 (Panel B). Yield was not altered by either storage method or sampling method. For both the sampling methods, the RIN was increased in the samples stored in RNAlater compared with snap frozen, and this was statistically significant (p < 0.05). FF, forceps, snap frozen; FRNA, forceps, RNAlater; SF, scalpel, snap frozen; SRNA, scalpel, RNAlater; RIN, RNA integrity number. Journal of Thoracic Oncology 2010 5, 956-963DOI: (10.1097/JTO.0b013e3181ddbbe9) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 Storage in RNAlater does not affect subsequent histology or immunohistochemistry assessment. Biopsies taken for tissue banking and stored in RNAlater were processed for routine histology and immunohistochemistry (Panels B and D). They were compared with sections from the same patients taken for diagnostic purposes (Panels A and C). (A, B) Hematoxylin and eosin-stained sections. (C, D) Immunostaining for pan-cytokeratin with MNF116 antibody. All images taken with a 100× magnification. Journal of Thoracic Oncology 2010 5, 956-963DOI: (10.1097/JTO.0b013e3181ddbbe9) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 5 RNA extracted from lung cancer biopsies is suitable for array analysis, but quality can influence results. Samples that met the predefined criteria as suitable for microarray analysis were hybridized to an Illumina Human HT12 v3.0 array. The resulting data met Illumina quality control standards. A, For GAPDH, the ratio of the normalized log intensities of the most 5′ probe compared with the most 3′ probe was not related to the quality of the RNA used (R2 = 0.04). However, for β tubulin (TUBB), the 5′:3′ ratio was weakly associated with RNA quality (R2 = 0.29, B). Journal of Thoracic Oncology 2010 5, 956-963DOI: (10.1097/JTO.0b013e3181ddbbe9) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions