Acharayothin Onchira, Thiengtrong Benjarat, Juengwiwattanakitti Panudeth, Anekwiang Panatna, Riansuwan Woramin, Chinswangwatanakul Vitoon, Tanjak Pariyada
Department of Surgery, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Siriraj Cancer Center, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Biopreserv Biobank. 2023 Feb;21(1):31-37. doi: 10.1089/bio.2021.0134. Epub 2022 Feb 25.
Colorectal cancer (CRC) is a common and lethal cancer worldwide. Extraction of high-quality RNA from CRC samples plays a key role in scientific research and translational medicine. Specimen collection and washing methods that do not compromise RNA quality or quantity are needed to ensure high quality specimens for gene expression analysis and other RNA-based downstream applications. We investigated the effect of tissue specimen collection and different preparation processes on the quality and quantity of RNA extracted from surgical CRC tissues. After surgical resection, tissues were harvested and prepared with various washing processes in a room adjacent to the operating room. One hundred fourteen tissues from 36 CRC patients were separately washed in either cold phosphate-buffered saline reagent ( = 34) or Dulbecco's modified Eagle's medium (DMEM; = 34) for 2-3 minutes until the stool was removed, and unwashed specimens served as controls ( = 34). Six tissue specimens were washed and immersed in DMEM for up to 1 hour at 4°C. Before RNA extraction, all specimens were kept in the stabilizing reagent for 3 months at -80°C. RNA was extracted, and the concentration per milligram of tissue was measured. RNA quality was assessed using the RNA integrity number (RIN) value. Different washing processes did not result in significant differences in RNA quantity or RIN values. In the six tissues that were washed and immersed in DMEM for 1 hour, RIN values significantly decreased. The quality of the extracted RNA from most specimens was excellent with the average RIN greater than 7. RNA is stable in specimens washed in different processes for short periods, but RIN values may decrease with prolonged wash times.
结直肠癌（CRC）是全球常见的致命性癌症。从CRC样本中提取高质量RNA在科学研究和转化医学中起着关键作用。需要采用不影响RNA质量或数量的标本采集和清洗方法，以确保获得用于基因表达分析和其他基于RNA的下游应用的高质量标本。我们研究了组织标本采集和不同制备过程对从手术切除的CRC组织中提取的RNA质量和数量的影响。手术切除后，在手术室相邻的房间里采集组织并采用各种清洗过程进行处理。来自36例CRC患者的114个组织分别在冷磷酸盐缓冲盐水试剂（n = 34）或杜尔贝科改良伊格尔培养基（DMEM；n = 34）中清洗2 - 3分钟，直至粪便清除，未清洗的标本作为对照（n = 34）。6个组织标本在4°C下用DMEM清洗并浸泡长达1小时。在提取RNA之前，所有标本在-80°C下保存在稳定试剂中3个月。提取RNA并测量每毫克组织的浓度。使用RNA完整性数（RIN）值评估RNA质量。不同的清洗过程在RNA数量或RIN值方面未导致显著差异。在6个用DMEM清洗并浸泡1小时的组织中，RIN值显著下降。大多数标本提取的RNA质量优异，平均RIN大于7。RNA在不同过程短时间清洗的标本中是稳定的，但RIN值可能会随着清洗时间延长而降低。
