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Gene expression studies using microarrays have provided important insights into understanding the mechanisms of transcriptional regulation in a variety of biological and disease phenomena. In a previous study, we developed Photo-DEAN, a universal-microarray-based RNA quantification method that enabled reverse transcription-free multiplex measurement of the absolute amount of RNA. Photo-DEAN promotes high-throughput and bias-less transcriptome analysis without the need for common controls or additional complicated normalization steps. In this study, we empirically identified two conditions (individual specificity and uniform duplex stability) necessary for in silico design of probe sequences, allowing the Photo-DEAN method to accurately measure the absolute amount of target RNA in total RNA. We then demonstrated that using the modified probe design conditions, the Photo-DEAN method successfully measured the absolute amount of pgi mRNA spiked into E. coli total RNA. The measurement was performed at five different sites in the coding region of pgi mRNA, exhibiting no significant site dependence. Theoretical considerations suggested that probe sequences longer than the previously used 30-bases better satisfy the necessary design conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We have developed a highly sensitive microarray-based method that determines the absolute amounts of mRNA in a total RNA sample in a multiplex manner without reverse transcription. This direct mRNA measurement promotes high-throughput testing and reduces bias in transcriptome analyses. Furthermore, quantification of the absolute amount of mRNA allows transcriptome analysis without common controls or additional, complicated normalization. The method, called Photo-DEAN, was validated using chemically synthesized RNAs of known quantities and mouse liver total RNA samples. We found that the absolute amounts of mRNA were successfully measured without the cDNA synthesis step, with a sensitivity of 15 zmol achieved in 7 h., (Copyright © 2017. Published by Elsevier Inc.)