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Messenger RNA expression analysis using quantitative real-time PCR Many mRNA and primer sequences have already been identified in pigs [20-22]. When genes were not described in this species, tBLASTn searches of the GenBank and PEDEblast ESTs databases, using known human and murine amino acid sequences, have been performed.These primers (purchased from Eurogentec) allowed the mRNA expression analysis of various genes involved in the innate immune response (Table 1). The qPCR was performed using cDNA synthesized as previously described [23]. Diluted cDNA (10X) was combined with primer/probe sets and IQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendations. The qPCR conditions were 98°C for 30 s, followed by 37 cycles with denaturation at 95°C for 15 s and annealing/elongation for 30 s (annealing temperature,Table 1). Real time assays were run on a Bio-Rad Chromo4 (Bio-Rad). The specificity of the qPCR reactions was assessed by analyzing the melting curves of the products and size verification of the amplicons. Each qPCR reaction included a reverse transcription negative control (RNA sample without reverse transcriptase) to check the absence of genomic DNA. To minimize sample variation, we used identical number of cells and high quality RNA. Samples were normalized internally using simultaneously the average cycle threshold (Cq) of Hypoxanthine PhosphoRibosyl-Transferase 1 (HPRT-1), Ribosomal Protein L 19 (RPL-19) and Tata Box Binding Protein 1 (TBP-1) [24] as references in each sample to avoid any artifact of variation in the target gene. These genes were selected as the reference genes because of their low variation between samples. A standard curve was generated using diluted cDNA. The correlation coefficients of the standard curves were > 0.995 and the concentrations of the test samples were calculated from the standard curves, according to the formula y = -M × Cq +B, where M is the slope of the curve, Cq the point during the exponential phase of amplification in which the fluorescent signal is first recorded as being statistically significant above background and B the y-axis intercept. Cq values were used to calculate the qPCR efficiency from the given slope according to the equation: qPCR efficiency = (10[-1/M] - 1) × 100. All qPCRs displayed efficiency between 90% and 110%. Expression data are expressed as relative values after Genex macro-analysis with three reference genes (Bio-Rad, Hercules, USA) [25]. |
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sunrq516516
金虫 (小有名气)
- 翻译EPI: 7
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爱与雨下: 金币+2 2014-04-07 14:43:23
868788: 金币+30, 翻译EPI+1, ★★★很有帮助 2014-04-07 15:11:03
爱与雨下: 金币+2 2014-04-07 14:43:23
868788: 金币+30, 翻译EPI+1, ★★★很有帮助 2014-04-07 15:11:03
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许多mRNA和引物序列已经在猪中被鉴定出来。当在无法在猪这个物种中找到（目标）基因时，tBLASTn利用已知的人类和鼠类的氨基酸序列查找了基因Bank和PEDEblast ESTs数据库。这些引物（从eruo...买的）可以分析多种参与先天免疫应答的基因的mRNA表达（表1）跟之前描述的一样（看文献23），qPCR是通过cDNA合成来实现的（呃，就是先合成cDNA，再PCR）。稀释后的cDNA（10倍）根据使用手册上建议的方法与引物/探针组以及SYBR Green(XXX)结合。qPCR的条件是98度30秒，接着是在37个循环（95度变性15秒-低温退火/延伸30秒（温度见表1））。实时分析是用bio-rad chromo4这个仪器来跑的。qPCR反应的特异性是通过产物的溶解曲线以及扩增子的大小来评估的。每个qPCR反应包括一个反转录阴性对照（RNA样品，不要放反转录酶），这是用来确认样品里没有DNA的。为了使样品的变动最小化，我们使用了等量的细胞数和高质量的RNA。样品是采用内标法来计算的，这里同时使用了HPRT-1，RPL-19和TBP-1的Cq作为内标（参考文献24），这样做是为了避免由对单个样品处理时人工操作而引起的对目标基因的误差。选择这些基因（作为内标）是因为文献中说它们在不同的样品中变化都比较小。标准曲线是根据cDNA的（梯度）稀释算出来的。标准曲线的相关系数大于0.995。待测样品的浓度是根据标准曲线来计算的，公式是y = -M × Cq +B。其中M是曲线仙侣，cq是在扩增的指数阶段，当荧光信号和背景产生显著统计差异时被记录下来的第一个点，B则是和Y轴的截距。cq值是根据已知的斜率用来计算qPCR的效率。等式是：qPCR efficiency= (10[-1/M] - 1) × 100.所有的qPCR的效率大约在90%到110%之间。用GeneX macro-analysis分析得到的基因表达结果是和三个内标基因比较的相对值。 呃……that's it.... |
2楼2014-04-07 14:41:29
sunrq516516
金虫 (小有名气)
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3楼2014-04-07 14:51:46
10