Hong Sang Hyun, Baek Hyun Ah, Jang Kyu Yun, Chung Myoung Ja, Moon Woo Sung, Kang Myoung Jae, Lee Dong Geun, Park Ho Sung
Chonbuk National University Hospital National Biobank of Korea, Jeonju, Korea.
J Korean Soc Coloproctol. 2010 Oct;26(5):316-23. doi: 10.3393/jksc.2010.26.5.316. Epub 2010 Oct 31.
The success of basic molecular research using biospecimens strongly depends on the quality of the specimen. In this study, we evaluated the effects of delayed freezing time on the stability of DNA and RNA in fresh frozen tissue from patients with colorectal cancer.
Tissues were frozen at 10, 30, 60, and 90 minutes after extirpation of colorectal cancer in 20 cases. Absorbance ratio of 260 to 280 nm (A(260)/A(280)) and agarose gel electrophoresis were evaluated. In addition, the RNA integrity number (RIN) was assayed for the analysis of the RNA integrity.
Regardless of delayed freezing time, all DNA and RNA samples revealed A(260)/A(280) ratios of more than 1.9, and all DNA samples showed a discrete, high-molecular-weight band on agarose gel electrophoresis. The RINs were 7.53 ± 2.04, 6.70 ± 1.88, 6.47 ± 2.58, and 4.22 ± 2.34 at 10, 30, 60, and 90 minutes, respectively. Though the concentration of RNA was not affected by delayed freezing, the RNA integrity was decreased with increasing delayed freezing time.
According to the RIN results, we recommend that the collection of colorectal cancer tissue should be done within 10 minutes for studies requiring RNA of high quality and within 30 minutes for usual RNA studies.
使用生物样本进行基础分子研究的成功与否在很大程度上取决于样本的质量。在本研究中，我们评估了延迟冷冻时间对结直肠癌患者新鲜冷冻组织中DNA和RNA稳定性的影响。
对20例结直肠癌患者的组织在切除后10、30、60和90分钟进行冷冻。评估260与280nm的吸光度比值（A(260)/A(280)）以及琼脂糖凝胶电泳情况。此外，检测RNA完整性数值（RIN）以分析RNA完整性。
无论延迟冷冻时间如何，所有DNA和RNA样本的A(260)/A(280)比值均超过1.9，并且所有DNA样本在琼脂糖凝胶电泳上均显示出一条离散的高分子量条带。在10、30、60和90分钟时，RIN值分别为7.53±2.04、6.70±1.88、6.47±2.58和4.22±2.34。虽然RNA浓度不受延迟冷冻的影响，但RNA完整性随延迟冷冻时间的增加而降低。
根据RIN结果，我们建议对于需要高质量RNA的研究，结直肠癌组织的采集应在10分钟内完成，对于常规RNA研究应在30分钟内完成。
