qPCR

定量即時聚合酶鏈反應(qPCR)利用螢光報告分子來量化擴增產物。與傳統 PCR 相同,該技術會對 DNA、cDNA 或 RNA 模板進行擴增,但在每個循環中,都會監測螢光訊號以進行相對或絕對定量。
此技術廣泛應用於多個研究領域,包括基因表達分析、基因分型、微RNA分析、基因變異分析及蛋白質分析。
精選類別
如何分析 qPCR 數據
透過測量並定量報告分子所發出的螢光;這些報告分子通常是能插入雙股 DNA 鹼基之間的染料(例如 SYBR® Green、溴化乙銨),或是專門設計用以結合 DNA 上特定序列的探針(例如 分子信標、TaqMan® 探針)。
qPCR 數據的相對定量
qPCR 數據主要有兩種定量方法。相對定量是較常見的方法,其利用 ΔΔCt 資訊,透過與對照基因比較,來確定樣本中目標基因的表達量或豐度比,並以參考基因的表達比進行歸一化。由於目標基因與參考基因的效率 E 值不同,此方法亦會考量 E 值之間的差異。此方法在基因表達分析中較為常用。
qPCR 數據的絕對定量
第二種方法——絕對定量——在環境微生物學中較為常見,並採用標準曲線(SC)法。 標準曲線法利用已知模板濃度 N0 的稀釋系列,透過 log(N0) 與 CT(閾值週期)的線性回歸來建立標準曲線。隨後利用此曲線計算樣本的模板濃度。此方法的基礎在於假設樣本的效率值與標準品的效率值相同。
相關文章
- After a traditional PCR has been completed, the PCR/qPCR data analysis is conducted by resolution through an agarose gel or, more recently, through a capillary.
- Quantitative PCR detection builds on basic PCR techniques and allows researchers to estimate the quantity of starting material in a sample with a much wider dynamic range.
- PCR assay guide navigates you through primer validation and other assay optimization factors to ensure high sensitivity and specificity for optimum DNA/ RNA quantification.
- PCR workflow's variability influenced by sample source, reverse transcription, and assay design, impacting reproducibility.
- The purpose of Hot Start PCR is to inhibit the PCR reaction in order to reduce nonspecific amplification, prevent the formation of primer dimers, and increase product yields.
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相關協定
- Our SYBR Green qPCR Protocol is a method designed to detect accurate quantification of gene expression and RT-PCR reactions.
- Chromatin Immunoprecipitation qPCR for studying gene regulation across conditions.
- Primer Concentration Optimization Protocol creates reaction matrix for testing primer concentrations against various partners.
- Optimization of qPCR conditions is important for the development of a robust assay. The two main approaches are optimization of primer concentration and/or annealing temperatures.
- Multiplex qPCR employing probe-based chemistries is a demanding application that often requires extensive optimization and validation.
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