Kenji Amemiya1, Yosuke Hirotsu2, Toshio Oyama3, Masao Omata4. 1. Genome Analysis Center, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi 400-8506, Japan. 2. Genome Analysis Center, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi 400-8506, Japan. Electronic address: hirotsu-bdyu@ych.pref.yamanashi.jp. 3. Department of Pathology, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi 400-8506, Japan. 4. Genome Analysis Center, Yamanashi Central Hospital, 1-1-1 Fujimi, Kofu, Yamanashi 400-8506, Japan; The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan.
Abstract
BACKGROUND: Tumor genetic alterations are determined to aid in selecting therapy and predicting prognosis. In routine clinical practice, targeted sequencing analysis is performed using formalin-fixed paraffin embedded (FFPE) tissues. However, successful genetic analysis remains challenging because FFPE DNA is fragmented during the sample preparation process. METHODS: Real-time PCR was performed to assess DNA quality and quantities. Targeted sequencing was performed using FFPE tissues fixed with different types of formalin. RESULTS: DNA was less fragmented from samples fixed in low formalin concentration (10% vs. 20%) and neutral buffered conditions (neutral buffered vs. non-neutral). DNA fragmentation increased over the fixation time. In a preliminary test study, we compared fixation using 10% neutral buffered formalin (n = 180) and 20% formalin (n = 26). The success rate of targeted analysis was higher using 10% neutral formalin (98.3%; 177/180) compared with 20% formalin (34.6%; 9/26). In a validation study with additional formalin-fixed paraffin embedded tissues fixed with 10% neutral buffered formalin (n = 860), we reproduced these results and achieved a high success rate for targeted sequencing analysis (98.4%; 846/860). CONCLUSION: Our data show that 10% neutral buffered formalin is recommended for fixation of formalin-fixed paraffin embedded samples to achieve high success rate of targeted sequencing analysis.
BACKGROUND: Tumor genetic alterations are determined to aid in selecting therapy and predicting prognosis. In routine clinical practice, targeted sequencing analysis is performed using formalin-fixed paraffin embedded (FFPE) tissues. However, successful genetic analysis remains challenging because FFPE DNA is fragmented during the sample preparation process. METHODS: Real-time PCR was performed to assess DNA quality and quantities. Targeted sequencing was performed using FFPE tissues fixed with different types of formalin. RESULTS: DNA was less fragmented from samples fixed in low formalin concentration (10% vs. 20%) and neutral buffered conditions (neutral buffered vs. non-neutral). DNA fragmentation increased over the fixation time. In a preliminary test study, we compared fixation using 10% neutral buffered formalin (n = 180) and 20% formalin (n = 26). The success rate of targeted analysis was higher using 10% neutral formalin (98.3%; 177/180) compared with 20% formalin (34.6%; 9/26). In a validation study with additional formalin-fixed paraffin embedded tissues fixed with 10% neutral buffered formalin (n = 860), we reproduced these results and achieved a high success rate for targeted sequencing analysis (98.4%; 846/860). CONCLUSION: Our data show that 10% neutral buffered formalin is recommended for fixation of formalin-fixed paraffin embedded samples to achieve high success rate of targeted sequencing analysis.
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