LPS 相关急性肺部炎症模型（免疫研究参考）
文献记录肺部炎症细胞反应，以及细胞因子和趋化因子表达变化。
Study design
以预先生成的 cDNA 为输入的 SYBR Green 实时定量 PCR 检测。每孔加入 10 ng cDNA，技术三重复；使用 PowerUp SYBR Green Master Mix 与 5 µM 正反向引物；按源方案循环条件扩增并读取 CT，以看家基因和参考实验组进行 ΔΔCT 分析。具体基因、引物和生物学分组按各研究设计确定。
Outcome measures
各样品目标序列的 CT 值；以看家基因归一化并以参考实验组计算 ΔΔCT/相对表达量。本方案不包含具体基因或疾病特异结论，输出判读依赖所选的引物、看家基因和实验分组。
Experimental animals
- Tissue
- lung
Experimental procedures
qPCR 反应体系 **Option 2****: SYBR qPCR** qPCR 反应体系 Prepare SYBR Master mix (ul per well): 1. 5ul PowerUp SYBR Green Master Mix (#A25776, Applied Biosystem-ThermoFisher) 1. 1ul Primers (Forward and Reverse, diluted in Nuclease-free water at 5µM) qPCR 反应体系 Add 6ul of SYBR Master mix to each well with an automatic pipette (Multipette E3, Eppendorf) qPCR 反应体系 Add 4ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics) 热循环 Perform PCR using the following cycling conditions in a LightCycler® 480 System (Roche): 1. 50˚C for 2min 1. 95˚C for 2min 1. 95˚C for 15s + 60˚C for 1min (x40cycles) 数据分析 Using the LightCycler Software obtain the threshold cycles (CT) signals from each samples. 数据分析 Perform ΔΔCT-method to analyze the expression data using the endogenous control genes and the reference experimental group.
Model identity
- Model name
- LPS 相关急性肺部炎症模型（免疫研究参考）
- Species
- Mus musculus
Biological characteristics
- Research questions
- 天然免疫应答及肺部炎症调控研究。
- Limitations
- 文献参考模型；LPS 炎症刺激不等同于活病原感染，不能代表传染病造模、病原传播或疫苗评价服务；不声明公司实际保有或已验证。
Experimental methods
Experimental protocols
- cDNA 模板的实时定量 PCR（qPCR，SYBR Green 分支）
This protocol adopts the SYBR Green branch of the protocols.io qPCR v1 record. The input is pre-generated cDNA (10 ng per well); RNA extraction and reverse transcription are not included. PowerUp SYBR Green Master Mix and forward/reverse primers are used on a LightCycler 480 with 50˚C for 2 min, 95˚C for 2 min, and 40 cycles of 95˚C for 15 s plus 60˚C for 1 min. CT values are read and analysed by the ΔΔCT method. No specific genes, primer sequences or disease-model readouts are asserted.
Reagents and materials
- SYBR Green qPCR Master Mix
SYBR Green 实时 qPCR 扩增
- qPCR 正反向引物（5 µM）
SYBR Green qPCR 扩增引物
- 无核酸酶水（Nuclease-free water）
引物稀释
References
- Johnston et al., Toxicol Sci 1998; PMID:10048133; DOI:10.1006/toxs.1998.2557
- Miquel Vila. Gene expression analysis by quantitative Real-Time PCR (qPCR). protocols.io. https://doi.org/10.17504/protocols.io.ewov1oyz2lr2/v1
- Bustin SA, et al. MIQE précis: Practical implementation of minimum standard guidelines for quantitative real-time PCR experiments. BMC Molecular Biology 2010;11:74. PMID 20858237. doi:10.1186/1471-2199-11-74
- ARRIVE 2.0 guidelines