Dedhia Pratiksha, Tarale Shivraj, Dhongde Gargi, Khadapkar Rashmi, Das Bibhu
Research and Development, SRL Ranbaxy Ltd., Andheri (E), Mumbai-400 093, India.
Asian Pac J Cancer Prev. 2007 Jan-Mar;8(1):55-9.
Formalin-fixed, paraffin-embedded (FFPE) tissues are the most invaluable source of diagnostic material for studying pathogenesis of cancer and a variety of other diseases. Unfortunately, DNA extracted from formalin fixed tissues is highly degraded due to cross-linking between nucleic acid strands. Real Time PCR has become the standard for gene copy as well as RNA transcript determination. Thus, optimum standardization of Real Time PCR is crucial for obtaining accurate quantification for both research as well as for clinical diagnosis. However there are various factors which have negative impact . The aim of our study was to establish a simpler method of extraction and Real Time PCR Optimization for FFPE extracted DNA. Five breast cancer tissues that were formalin fixed and paraffin embedded were used for DNA extraction with four different methods. Extracted DNA was amplified with different primer sets that gave amplimers of different size. Optimization of Real Time PCR for EMSY, cyclin D1 and beta-globin genes was carried out on DNA obtained using heat treatment protocol for annealing temperature, primer concentration and template concentration. Highest quantity of DNA was obtained without the use of expensive reagents and in short time frame. PCR positivity was observed in case of shorter amplimer up to 250 bp in length. Amplimers of higher length failed to amplify with paraffin extracted DNA. Optimum annealing temperature for EMSY, Cyclin D1 and beta-globin genes were 60 degrees C, 60 degrees C and 61 degrees C respectively. Good results were seen with a primer concentration of 300 nM and 5 ng of template DNA. This study indicates that DNA obtained from formalin fixed paraffin embedded tissue is highly fragmented and can be used for successful amplification of shorter amplification products up to 250 bp in length. Optimization of real time PCR is important, especially while using SYBR green dye chemistry.
福尔马林固定、石蜡包埋（FFPE）组织是研究癌症及多种其他疾病发病机制最宝贵的诊断材料来源。不幸的是，从福尔马林固定组织中提取的DNA由于核酸链之间的交联而高度降解。实时荧光定量PCR已成为基因拷贝数以及RNA转录本测定的标准方法。因此，实时荧光定量PCR的最佳标准化对于获得准确的定量结果以用于研究和临床诊断都至关重要。然而，有多种因素会产生负面影响。我们研究的目的是建立一种更简单的方法，用于从FFPE提取的DNA中进行提取和实时荧光定量PCR优化。使用四种不同方法对五个福尔马林固定、石蜡包埋的乳腺癌组织进行DNA提取。用不同的引物组对提取的DNA进行扩增，这些引物组产生不同大小的扩增子。针对EMSY、细胞周期蛋白D1和β-珠蛋白基因，对使用热处理方案获得的DNA在退火温度、引物浓度和模板浓度方面进行实时荧光定量PCR优化。在不使用昂贵试剂且短时间内获得了最高量的DNA。对于长度达250 bp的较短扩增子观察到PCR阳性。较长长度的扩增子未能用石蜡提取的DNA扩增。EMSY、细胞周期蛋白D1和β-珠蛋白基因的最佳退火温度分别为60℃、60℃和61℃。引物浓度为300 nM和模板DNA为5 ng时效果良好。本研究表明，从福尔马林固定石蜡包埋组织中获得的DNA高度片段化，可用于成功扩增长度达250 bp的较短扩增产物。实时荧光定量PCR的优化很重要，尤其是在使用SYBR Green染料化学方法时。
