Systematic selection of reference genes for normalization of circulating RNA transcripts in pregnant women based on RNA-seq data

Chim, S. S. C.; Wong, K. K. W.; Chung, C. Y. L.; Lam, S. K. W.; Kwok, J. S. L.; Lai, C.-Y.; Cheng, Y. K. Y.; Hui, A. S. Y.; Meng, M.; Chan, O.-K.; Tsui, S. K. W.; Lee, K.-Y.; Chan, T.-F.; Leung, T.-Y.

doi:10.1101/165654

bioRxiv · 10.1101/165654

Systematic selection of reference genes for normalization of circulating RNA transcripts in pregnant women based on RNA-seq data

Chim, S. S. C.·Wong, K. K. W.·Chung, C. Y. L.·Lam, S. K. W.·Kwok, J. S. L.·Lai, C.-Y.·Cheng, Y. K. Y.·Hui, A. S. Y.·Meng, M.·Chan, O.-K.·Tsui, S. K. W.·Lee, K.-Y.·Chan, T.-F.·Leung, T.-Y.

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Abstract

RNA transcripts circulating in peripheral blood represent an important source of non-invasive biomarkers. To accurately quantify the levels of a circulating transcript, one needs to normalize the data with internal control reference genes, which are detected at relatively constant levels across different blood samples. A few stably-expressed reference gene candidates have to be selected from transcriptome data before validation of their stable expression by reverse-transcription quantitative polymerase chain reaction. However, there is a lack of transcriptome, let alone whole-transcriptome, data from maternal blood. To overcome this shortfall, we performed RNA-seq on blood samples from women presented with preterm labor. Of 11215 exons detected in the maternal blood whole-transcriptome, we systematically identified a panel of 395 genes comprising exons that were detected at a coefficient of variation (CV) ranging from 7.75%-17.7%. Their levels were considerably less variable than any GAPDH exon (minimum CV, 27.3%). Upon validation, selected genes from this panel remained as more stably expressed than GAPDH in maternal blood. This panel is over-represented with genes involved with actin cytoskeleton, macromolecular complex and the integrin signaling pathway. This groundwork provides a starting point for systematically selecting reference gene candidates for normalizing the levels of circulating RNA transcripts in maternal blood.

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Chim, S. S. C., Wong, K. K. W., Chung, C. Y. L., Lam, S. K. W., Kwok, J. S. L., Lai, C.-Y., Cheng, Y. K. Y., Hui, A. S. Y., Meng, M., Chan, O.-K., Tsui, S. K. W., Lee, K.-Y., Chan, T.-F., Leung, T.-Y.. 2017-07-19. Systematic selection of reference genes for normalization of circulating RNA transcripts in pregnant women based on RNA-seq data. https://doi.org/10.1101/165654

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