Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies.

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies. Since SYBR Green I dye is a nonspecific intercalating dye, the reaction is made specific by using "hot-start" PCR and empirically determined annealing and signal acquisition temperatures for each gene-specific primer. Relative expression levels were quantified by constructing a standard curve using cDNA dilutions of a highly expressed gene. Using this approach, real-time PCR validated 17 of 21 (71%) genes identified by DNA arrays, and all but 1 of 13 (91%) genes identified by differential display PCR (DD-PCR). Validation of differentially expressed genes detected by array analysis was related to hybridization intensity. Real-time RT-PCR results suggest that genes identified by DNA arrays with a two to fourfold difference in expression cannot be accepted as true or false without validation. Validation of differentially expressed genes detected by DD-PCR was not affected by band intensities. Regardless of the gene expression profiling technology (microarrays, DD-PCR, serial analysis of gene expression and subtraction hybridization), once the sequence of gene of interest is known, the real-time RT-PCR approach is well suited for validation of differential expression since it is quantitative and rapid and requires 1000-fold less RNA than conventional assays.

Modified AFLP technique for rapid genetic characterization in plants.

The standard amplified fragment-length polymorphism (AFLP) technique was modified to develop a convenient and reliable technique for rapid genetic characterization of plants. Modifications included (i) using one restriction enzyme, one adapter molecule and primer, (ii) incorporating formamide to generate more intense and uniform bands and (iii) using agarose gel electrophoresis. Sea oats (Uniola paniculata L.), pickerel-weed (Pontederia cordata L.), Bermudagrass (Cynodon dactylon L.) and Penstemon heterophyllus Lindl. were used to determine the ability to generate adequate resolution power with both self- and cross-pollinated plant species including cultivars, ecotypes and individuals within populations. Reproducibility of bands was higher in all the AFLP experiments compared to random amplified polymorphic DNA (RAPD). Formamide with or without bovine serum albumin improved band intensities compared to dimethyl sulfoxide and the standard reaction mixture with no organic solvents. Comparison between RAPD and modified AFLP using sea-oats population samples proved that modified AFLP exhibits (i) a low number of faint bands with increased specificity of amplified bands, (ii) a significantly higher number of polymorphic loci per primer, (iii) less primer screening time, (iv) easy scoring associated with fewer faint bands and (v) greatly enhanced reproducibility. The technique described here can be applied with a high degree of accuracy for plant genetic characterization.