Green Tina Marie, de Stricker Karin, Møller Michael Boe
Department of Pathology, Odense University Hospital, Odense, Denmark.
Diagn Mol Pathol. 2009 Dec;18(4):243-9. doi: 10.1097/PDM.0b013e3181a06f42.
Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral oncogene homolog 1 gene were also highly ranked by both programs. In addition, we measured the relative expressions of 7 target genes by Q-RT-PCR, using PCR primer-probes with amplicon sizes up to 105 bases. The correlation coefficient for expression measured in matched frozen and paraffin-embedded samples was 0.93 (P<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.
将定量逆转录聚合酶链反应（Q-RT-PCR）数据标准化到合适的组织特异性参考基因是解释结果的重要组成部分。本研究旨在通过使用两种统计软件程序geNorm和NormFinder，确定用于标准化以非肿瘤性淋巴结和弥漫性大B细胞淋巴瘤为代表的淋巴组织中基因表达的最合适参考基因。此外，我们想通过研究配对的冷冻和石蜡包埋样本中相关靶基因的基因表达，来验证石蜡包埋样本在Q-RT-PCR研究中的实用性。此外，我们研究了扩增子大小对石蜡包埋组织中Q-RT-PCR效率的影响。对6个假定的参考基因在21对速冻和福尔马林固定、石蜡包埋的淋巴结和淋巴瘤中进行表达稳定性测试。根据它们作为参考基因的适用性对这些基因进行排名。根据两种统计方法，β-葡萄糖醛酸酶在速冻和石蜡包埋样本中都是最合适的单一参考基因。TATA盒结合蛋白基因和阿贝尔逊鼠白血病病毒癌基因同源物1基因在两个程序中也都排名很高。此外，我们使用扩增子大小达105个碱基的PCR引物探针，通过Q-RT-PCR测量了7个靶基因的相对表达。在用合适的参考基因标准化后，配对的冷冻和石蜡包埋样本中测量的表达的相关系数为0.93（P<0.01）。因此，我们表明，当使用经过充分验证的参考基因时，福尔马林固定、石蜡包埋的淋巴样样本适用于Q-RT-PCR。
