1 / 30

Real-Time PCR 实验

Real-Time PCR 实验. 医学研究中心 郭卫. 目标. 建立 SYBR GREEN 实时定量 PCR 体系 掌握绝对定量和相对定量的原理和应用 运用 Bio-Rad IQ2 PCR 仪进行实验的设定和分析 了解定量 PCR 实验的操作注意事项. SYBR GREEN 实时定量 PCR 体系. 反应体系的建立及优化 : SYBR Green 使用浓度 : 太高抑制 Taq 酶活性，太低，荧光信号太弱，不易检测 MgCl 2 的浓度 : 可以降低以减少非特异性产物 反应 Buffer 体系的优化 ---- 使用 REAL-TIME PCR KIT

selina
Télécharger la présentation

Real-Time PCR 实验

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Real-Time PCR实验 医学研究中心 郭卫

  2. 目标 建立SYBR GREEN实时定量PCR体系 掌握绝对定量和相对定量的原理和应用 运用Bio-Rad IQ2 PCR仪进行实验的设定和分析 了解定量PCR实验的操作注意事项

  3. SYBR GREEN实时定量PCR体系 反应体系的建立及优化: • SYBR Green 使用浓度:太高抑制Taq酶活性，太低，荧光信号太弱，不易检测 • MgCl2的浓度:可以降低以减少非特异性产物 • 反应Buffer 体系的优化 ----使用REAL-TIME PCR KIT • Primer：引物的特异性高，否则扩增有杂带，定量不准 • 反应温度和时间参数:由酶和引物决定 • 其他与常规PCR相同

  4. SYBR GREEN法引物设计 • 扩增子的长度应不超过400bp，理想的最好能在80－150bp • 引物长度18~25bp，Tm值在63-67 ℃，GC含量30-80%（最好40-60 %），退火温度在58-62 ℃ • 上下游引物间Tm值差小于4 ℃ • 避免引物错误引发，且引物末端（最后5个核苷酸)不能超过2个G或C • 避免引物内和引物间的互补（避免形成发夹结构和引物二聚体） • 跨一个或多个内含子设计引物（避免对基因组DNA的扩增） • 使用Blast检查引物特异性（www.nibi.nlm.nih.gov/blast)

  5. 跨内含子设计引物

  6. n PCR的理论方程：Y=x×(1+ Ev) Y：扩增物数量； X ：起始模板数量；Ev：扩增效率；n：扩增循环数 Threshold 阈值 荧光超过本底，进入相对稳定对数增长期时的临界数值 Baseline 背景曲线的一段，范围 CT值 threshold Cycle，阈值与扩增曲线相交，所得交点所对应的循环数。

  7. 为什么CT ∝起始DNA浓度?

  8. 为什么CT ∝起始DNA浓度?

  9. 绝对定量和相对定量

  10. 绝对定量 • 由已知模板的浓度值和CT值标准曲线 • 获得未知样品的模板浓度 • 标准品： • 必须与目标基因引物相同，且扩增效率一致 • 标准品必须准确定量 (e.g. UV spectrophotometer) • 每套实验必须设定同样的域值，才可计算未知样品的Ct值

  11. Gene of Interest (Target) BCL-2: Proto-oncogene which inhibits apoptosis via the regulation of reactive oxygen species. Over-expression of BCL-2 is a common cause of multi-drug resistance. Normalized Fluorescence Real Time Amplification Curves Standard Curve Numerical Data Ct Values GAPDH Efficiency = 10 -1/M(slope) -1 Standards Housekeeping Gene (Reference) GAPDH: An enzyme involved in glycolysis. The GAPDH gene is constitutively expressed at high levels in almost all tissues. Ct values Threshold Calculated Concentrations BCL-2 Four samples of human genomic DNA were used. One of these samples was repeated and designated as the calibrator. Samples Experiment Example

  12. 相对定量——基因表达量的比较 • 使用∆∆CT分析法或Pfaffel法 • 样品模板必须用内参照基因标准化 • 运用最广泛和最强大的方法

  13. Quantitation Analysis Formula = 2 –DD Ct Subtract the mean Ct Value for Housekeeping Gene from Gene of Interest for each concentration Determine the log For each concentration Delta-Delta Ct: Delta Ct Calibrator – (minus) Delta Ct Sample Relative Value: 2 to the power of negative Delta-Delta Ct Determine the mean Ct value for each sample (Gene of Interest and Housekeeper) Are these Efficiencies Truly Equal ? Delta Ct Calibrator: Mean Ct Gene of Interest - (minus) Mean Ct Housekeeping gene Delta Ct Sample: Mean Ct Gene of Interest – (minus) Mean Ct Housekeeping gene [MeanCt Gene of Interest (Calibrator) – MeanCt Housekeeping Gene (Calibrator)] Minus [MeanCt Gene of Interest (Sample) – MeanCt Housekeeping Gene (Sample)] Determine the mean Ct values for Calibrator (Gene of Interest), Sample (Gene of Interest), Calibrator (Housekeeping gene), And Sample (Housekeeping gene) Draw a line of best fit And determine the Equation of the line Mean Ct values for Each sample (Gene of Interest and Housekeeper) - Gene of Interest E = 95 % Plot the Ct difference With Log concentration 2 Gradient of the line must Be less than 0.1 Only one set of standard curves Is run for each gene. However the Efficiencies must be the same. Housekeeper E = 97 % Comparative Delta-Delta Ct

  14. Formula = D CPTarget (control – sample) E Target Delta Cp Target: Calibrator (Gene of Interest) Minus Sample (Gene of Interest) Delta Cp Reference: Calibrator (Housekeeper) Minus Sample (Housekeeper) Efficiency for Gene of Interest to the power Of Delta Cp Target Efficiency for House- Keeping gene to the Power of Delta Cp Reference Efficiencies are determined From the Standard curves For Gene of Interest and Housekeeping gene Determine the mean Ct of both Calibrator and Sample for both Gene of Interest and Housekeeping gene Housekeeping Gene E = 97 %; A = 1.97 D CP Reference (control – sample) Ratio of Gene of Interest To Housekeeping gene (Divide the last to sets Of numbers) EReference Only one set of standard Curves is run for each Gene and Efficiencies Do not need to be the same Gene of Interest E = 110%; A = 2.10 Pfaffel (2001)

  15. 注意事项：

  16. 选择一个内标基因。 • 确定内标的有效性，确保它不会受到实验处理的影响。 • 通过PCR 扩增目标基因和内标基因RNA 或cDNA 的一系列梯度稀释模板确保它们的扩增效率相同。 • 最後通过2－△CT 计算将统计数据转化成线性形式而不是原始CT值

  17. Precision is essential to Real-Time PCR! What factors affect precision? • 移液器 - 经常校准移液器 - 确定移液器和枪头配套 • 分装试剂 - 缓慢移液 - 用新枪头 - 靠近液面取液 - 尽可能使用自动移液装置 • 温度 - 仪器孔与孔之间，设定温度与实际温度之间差异要小 • 平衡时间 - 所有的孔达到相同温度的时间 - 收集所有的孔数据的时间 • 光源强度一致 - 光源照射每个样品的强度达到孔与孔信号恒定 • 必要的维护 - 仪器定期维护

  18. 本次实验体系 模板（Template） 2ul 引物（Primer） 4ul PCR试剂（Mix） 10ul 水 4ul 总量 20ul

  19. 实 验 报 告 实验名称 专业班级姓 名学 号 一、实验目的与原理（10分） 二、实验仪器及样本（10） 三、实验步骤（20分） 四、实验结果与讨论（30分+30分）

More Related