A novel procedure for absolute real-time quantification of gene expression patterns

We propose a solution here with a novel procedure, which may accurately quantify the total cDNA conventionally prepared from a biological sample at the resolution of ~70 pg/μl, and reliably estimate the absolute numbers of transcripts in a picogram of cDNA. In comparison to the relative quantification, cDNA-based absolute (CBA) qPCR method is found to be more sensitive to gene expression variations caused by factors such as developmental and environmental variations. If the number of target transcript copies is further normalized by reference transcripts, cell-level variation pattern of the target gene expression may also be detectable during a developmental process, as observed here in cases across species (Ipomoea purpurea, Nicotiana benthamiana) and tissues (petals and leaves).By allowing direct comparisons of results across experiments, the new procedure opens a window to make inferences of gene expression patterns across a broad spectrum of living systems and tissues. Such comparisons are urgently needed for biological interpretations of gene expression variations in diverse cells.Being a key stage of functional realization of genome, gene expression has been increasingly quested for more details in various investigations [1-4]. Relative to still costly implementations of omics approaches, real-time quantitative polymerase chain reaction (qPCR) technique remains a top choice for comparison of gene expressions in cases of a small gene number but variable sample sizes because of the sensitivity of florophors, the efficiency of PCR [5], and the relatively low cost. Albeit having widespread usages in laboratories [6], qPCR may have some difficulties in interpretations of its results since gene expressions have been estimated as fold changes [7], which may be hard to compare across tissues and experiments without a common basis.Depending on the appropriate internal control (reference) genes, the relative qPCR method estimates fold change of expression difference bet