RT-PCR and allele-specific cDNA quantification analysis with the WAVE dHPLC system. The parental alleles of the ZmNAC1 gene were cloned and sequenced, and an allelic polymorphism that had 4 bp less in the 5′UTR region of 87-1 than Zong3 was found. RT-PCR was then performed using primers designed for the conserved region between the alleles that encompassed the 4 bp Indel region. The RT-PCR products were separated and quantified using a WAVE dHPLC system. The longer DNA fragments that corresponded to the Zong3 allele had a higher affinity and therefore took a longer time to be eluted from the WAVE column than the shorter DNA fragments of the 87-1 allele eluted earlier. The x-axis shows the time in minutes when the DNA fragments were eluted. The y-axis demonstrates the UV absorbance that was used to measure DNA concentration or expression level. This analysis quantifies the allele-specific transcript as a relative ratio and does not measure the absolute transcription level.
Li et al. BMC Plant Biology 2012 12:220 doi:10.1186/1471-2229-12-220