Impact of Thawing on Reference Gene Expression Stability in Renal Cell Carcinoma Samples
Impact of Thawing on Reference Gene Expression Stability in Renal Cell Carcinoma Samples
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DOI：
10.1097/pdm.0b013e31824d3435
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发表时间：
2012-09-01
影响因子：
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通讯作者：
Wang, LiMin
中科院分区：
文献类型：
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作者：
Ma, Yi;Dai, HuiLi;Wang, LiMin
越来越多的样本是从生物库中获得的，用于生物医学研究；然而，这些样本中的一些可能会在加工之前进行解冻。我们的目的是评估参考基因在解冻后的肾癌样本中的表达稳定性。获得16份配对的恶性和非恶性肾组织标本，每个标本被分成4份样品，然后快速冷冻并在-80℃下保存。通过实时定量聚合酶链式反应，对解冻后的组织进行时程研究，以评估肾癌样品中最常用的10个参考基因：ACTB、ALAS1、B2M、GAPDH、HMBS、HPRT、PIA、RPLP0、TBP和Tubb的表达稳定性。根据geNorm M值，PPIA在0、15和30分钟时间点是最稳定的基因(M分别为0.82、0.85和0.76)，而GAPDH在5、15和30分钟时间点排名最后(M分别为1.38、1.44和1.39)。解冻时间与2与所有候选参考基因的交叉点值的幂呈线性回归关系(P<；0.05)。所有参比基因的平均变异系数在5、15和30min较0min显著增加(P<；0.01)。总之，使用geNorm算法，PPIA被确定为在解冻相似时间段的恶性和非恶性肾组织样本中最稳定表达的基因。所有参考基因都随着解冻时间的延长表现出较高的变异，在比较不同解冻时间段的样品时，建议使用多个候选参考基因的组合。
More and more samples are obtained from biobanks for biomedical research; however, some of these samples may undergo thawing before processing. We aim to evaluate the reference gene expression stability in thawed renal cell carcinoma samples. Sixteen matched malignant and nonmalignant renal tissue samples were obtained and each sample was divided into 4 aliquots before being snap frozen and stored at -80 degrees C. By quantitative real-time polymerase chain reaction, a time-course study was conducted on the thawed tissue to evaluate the expression stability of a panel of the 10 most frequently used reference genes in renal cell carcinom samples: ACTB, ALAS1, B2M, GAPDH, HMBS, HPRT, PPIA, RPLP0, TBP, and TUBB. As shown by geNorm M values, PPIA was the most stable gene at the 0-, 15-, and 30-minute time points (M = 0.82, 0.85, and 0.76, respectively), whereas GAPDH was ranked last at the 5-, 15-, and 30-minute time points (M = 1.38, 1.44, and 1.39, respectively). A positive correlation was found by linear regression between the thawing time and 2 to the power of crossing point values of all candidate reference genes (P < 0.05). The mean coefficient of variance of all reference genes increased significantly at time points 5, 15, and 30 minutes compared with 0 minutes (P < 0.01). In conclusion, using the geNorm algorithm, PPIA was identified as the most stably expressed gene between malignant and nonmalignant renal tissue samples that were thawed for similar time periods. All the reference genes showed high variations along with the thawing time; it should be recommended to use a combination of several candidate reference genes when comparing samples thawed for different time periods.