【原】长期神经病理性疼痛行为学改变与突触可塑性和边缘回路改变的相关性研究：一项小鼠的对照观察研究（十）

 英语晨读 ·

山东省立医院疼痛科英语晨读已经坚持10余年的时间了，每天交班前15分钟都会精选一篇英文文献进行阅读和翻译。一是可以保持工作后的英语阅读习惯，二是可以学习前沿的疼痛相关知识。我们会将晨读内容与大家分享，助力疼痛学习。

本次文献选自Francesca Guidaa，Monica Iannottaa，Gabriella Misso, et al. Pain. 2022.本次学习由李芸主治医师主讲。

2.8. Gene expression analysis

2.8.1. RNA extraction and cDNA synthesis

Total RNA was isolated from either hippocampus or prefrontal cortex tissues of 4 groups of samples including the 12-month sham, 1-month sham, 12-month SNI, and 1-month SNI mice. TRIzol Reagent (Invitrogen, Carlsbad, CA) was used according to manufacturer's protocol. RNA was finally eluted into 20 mL of 0.1 mM EDTA buffer to concentrate the extract. RNA purity and quantity were measured by NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, NC) using the 260/280 ratio. RNA samples were stored at -80˚C until further processing. The cDNA synthesis was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA).

2.8. 基因表达分析

2.8.1. RNA提取和cDNA合成

从4组样本的海马体或前额叶皮层组织中分离总RNA，包括12个月的假手术组、1个月的假手术组、12个月的SNI组和1个月的SNI组。根据制造商的方案，使用TRIzol试剂。RNA最后被洗脱到20mL的0.1mM EDTA缓冲液中，以浓缩提取物。用NanoDrop ND-1000分光光度计测量RNA的纯度和数量，使用260/280比率。RNA样品在-80˚C下保存，直到进一步处理。使用高容量cDNA反转录试剂盒进行cDNA合成。

2.8.2. Gene expression array

Equal amounts of cDNA from each group of mice were mixed to obtain 4 pools in duplicate for both the brain areas. In detail, each pool included 5 cDNA samples from either the 1-month mice or the 12-month mice, both for sham and SNI mice. The gene expression profiling was performed using TaqMan Array Mouse Immune Panel (Applied Biosystems), which allows simultaneous detection of 96 genes, including cytokines, chemokines, growth factors, and other immune response genes, and TaqMan Universal PCR Master Mix (Applied Biosystems) following manufacturer’s manuals. The Ct values were determined using ViiA 7 software (Applied Biosystems) and setting a threshold of 0.2. The Ct values above 32.0 were set as undetermined. The relative quantification of each mRNA was calculated with the ΔΔCt method and fold change (FC) with the equation 2-ΔΔCt.

2.8.2. 基因表达阵列

来自每组小鼠的等量cDNA被混合，以获得两个脑区的4个库。详细地说，每个库包括5个cDNA样本，分别来自1个月的小鼠或12个月的小鼠，包括假手术组和SNI组。基因表达分析使用TaqMan阵列小鼠免疫面板（Applied Biosystems）进行，该面板可以同时检测96个基因，包括细胞因子、趋化因子、生长因子和其他免疫反应基因，TaqMan通用PCR Master Mix（Applied Biosystems）遵循制造商的方案。使用ViiA 7软件（Applied Biosystems）确定Ct值，并设定阈值为0.2。高于32.0的Ct值被设定为待定。用ΔΔCt法计算每个mRNA的相对定量，用公式2-ΔΔCt计算折叠变化（FC）。