RT-PCR-设计、计算与统计方法 Real-Time Quantitative RT-PCR Design, Calculations, and Statistics.pdf

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online reduces the time to publication by several weeks. LETTER TO THE EDITOR Real-Time Quantitative RT-PCR: Design, Calculations, and Statistics Two recent letters to the editor of The Plant a design was investigated by Hellemans early phase, the progress of this curve Cell (Gutierrez et al., 2008; Udvardi et al., et al. (2007) where they compared gene being determined by the amplification effi- 2008) highlighted the importance of follow- maximization to sample maximization; but ciency, E. The basic formula applying to ing correct experimental protocol in quan- to enable effective statistical comparison of qRT-PCR aims to convert the number of titative RT-PCR (qRT-PCR). In these letters, treatments, a strategy that may be termed cycles at a threshold level of fluorescence the authors outlined measures to allow ‘‘treatment maximization’’ is required. As a (more generally termed the quantification precise estimation of gene expression by plate can be viewed as a statistical block, cycle or Cq; Hellemans et al., 2007) into a ensuring the quality of material, refining the best option would be to separate relative quantity of input template present laboratory practice, and using a normaliza- complete biological replicates (i.e., one at the start of the PCR. If we take the tion of relative quantities of transcripts of biological sample of each treatment)