英语翻译RNA Extraction and cDNA SynthesisTotal RNA was extracted

英语翻译
RNA Extraction and cDNA Synthesis
Total RNA was extracted from homogenized XX tissue by using the TRIzol method (Invitrogen,Karlsruhe,Germany).The tissue was squeezed by using a Dounce homogenizator (Sartorius,Goettingen,Germany) with 1000 mL TRIzol reagent.The homogenate was separated in aqueous and organic phases by the addition of 200 mL chloroform followed by centrifugation at 12,000g for 15 minutes at 4℃.RNA was precipitated by addition of 500 mL isopropanol and centrifugation at 12,000g for 10 minutes at 4℃.The resulting RNA was washed in 70% ethanol and solubilized in diethyl pyrocarbonate–treated water.After elution,final RNA concentrations were determined photometrically.Complementary cDNA was synthesized from 1–13 mL of RNA (solution concentration,1 mg/mL) by reverse transcriptase reaction by using 1 mmol/L oligo-dT primers and 200 U of M-MLV reverse transcriptase(Promega,Mannheim,Germany) at 42℃ for 1 hour.
Real-time Polymerase Chain Reaction
Polymerase chain reaction (PCR) was performed as described(24) by using a real-time (RT) PCR cycler (Rotor-Gene 2000; Corbett Research,Sydney,Australia).Briefly,for each sample to be investigated,160 ng of cDNA template was added to 10 mL of QuantiTect SYBRgreen master mix (Qiagen) and 4 mL of both forward and backward primers (short primers’ final concentration,0.6 mmol/L each; Table 1) to a total volume of 20 mL with RNA-free water.PCR results were quantified by using standard curves generated from external standard templates.The cDNA for external standards was generated by nested RT-PCR (long primers; Table 1) from total RNA obtained by stimulation of HaCaT cell culture with microbial LPS.Amplification products were purified by using MinElute PCR Purification Kit (Qiagen),and copies per mL were calculated according to the formula,molecules/mL = DNA [g/mL]/DNA-length [base pairs _660] _ 6.022 _ 1023.Copy numbers from 1 _ 102 to 1 _ 1011 were then submitted to RT-PCR (short primers; Table 1) to generate standard curves.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (housekeeping gene) as well as the proinflammatory cytokines tumor necrosis factor–alpha (TNF-a) and interleukin-1beta (IL-1b) were run in parallel with all experimental samples.Cycling conditions were 95℃ for 15 minutes (initial denaturation) followed by 35 cycles of 94℃ for 15 seconds (denaturation),56℃ for 30 seconds (annealing),and 72℃ for 75 seconds (extension).The number of copies was extrapolated from the corresponding standard curve and set relative to 1 mg of RNA introduced into the RT reaction and normalized to GAPDH.PCR products were separated and analyzed to control for single amplification products and of resulting product size by agarose gel(2%) electrophoresis (25).Specificity of amplification reaction was further controlled by melting curves,no template controls,and no RT controls.

核酸提取和合成
总核糖核酸提取匀浆某某组织使用的提取方法（公司,卡尔斯鲁厄,德国）.该组织被挤压,使用douncehomogenizator（缝纫机,德国哥廷根,）1000毫升Trizol试剂.匀浆分离水相和有机相的增加200毫升氯仿后离心15分钟,在4℃12000g.核糖核酸沉淀加入500毫升的异丙醇和离心12000g10分钟在4℃.由此产生的核酸洗用70%乙醇溶解在二乙基焦碳–处理水.洗脱后,最后的核糖核酸浓度测定光度法.互补基因合成核糖核酸（1–13毫升溶液的浓度,1毫克/毫升）的逆转录反应使用1毫摩尔/升寡核苷酸引物和200的反转录酶逆转录酶（试剂,曼海姆,德国）在42℃1小时.
实时聚合酶链反应
聚合酶链反应（聚合酶链反应）进行描述（24）采用实时（逆转录）聚合酶链反应循环（rotor-gene2000；科贝特研究,悉尼,澳大利亚）.简单地说,每个样本进行调查,160毫微克的基因模板,加入10毫升quantitectsybrgreen混合大师（试剂盒）和4毫升的正向和反向引物（短引物的浓度,0.6毫摩尔/升每；表1）,总体积20毫升与RNA的免费水.反应的结果是用量化的标准曲线产生的外部标准模板.基因为外部标准产生的巢式RT - PCR（长引物；表1）从总得到刺激角质形成细胞培养和微生物毒素.扩增产物进行纯化,采用聚合酶链反应（minelute试剂盒纯化试剂盒）,并拷贝/毫升按公式计算,分子/毫升=NA[克/毫升]/[碱基对dna-length_660]_6.022_1023.拷贝数量从1102到11011__然后提交给RT - PCR（短引物；表1）生成标准曲线.3 -磷酸甘油醛脱氢酶（型）（看家基因）以及炎性细胞因子肿瘤坏死因子（TNF -α–）和白细胞（基因）是并行运行的所有实验样品.循环条件95℃15分钟（初始变性）其次是35周期的94℃15秒（变性）,56℃30秒（退火）,和72℃75秒（扩展）.份数推断相应的标准曲线和相对的1毫克的核糖核酸引入反应和正常型.聚合酶链反应产物进行分离和分析,为单片机控制的扩增产物和由此产生的产品大小的琼脂糖凝胶电泳（2%）（25）.特异性扩增反应进一步控制熔化曲线,没有模板控制,并没有实时控制.