New method of measuring absolute RNA copy numbers redefines our ability to perform quantitative biology

Transcript regulation is essential for cell function, and misregulation can lead to disease. Despite technologies to survey the transcriptome, we lack a comprehensive understanding of transcript kinetics, which limits quantitative biology. This is an acute challenge in embryonic development, where rapid changes in gene expression dictate cell fate decisions.

Now, researchers at the Francis Crick Institute have devloped a method, using spike-ins to calibrate transcript reads, to measure absolute mRNA transcript numbers and calculate transcriptome kinetics, in some cases, in kilobases per minute per allele.

To calculate absolute transcript numbers and kinetics, the researchers collected Xenopus tropicalis eggs (time 0) and then synchronously developing embryos over a time course. After embryo homogenization and prior to RNA extraction, they added a known amount of spike-in RNAs per embryo to each sample independently. This ensured that RNA standards underwent the same variations in recovery as the endogenous embryonic transcripts during RNA purification, library preparation, and sequencing. Spike-ins included 92 synthetic RNAs available from the External RNA Control Consortium (ERCC) and three of the E. coli-derived ArrayControl spike-ins.


By ultra-high-frequency sampling of Xenopus embryos and absolute normalization of sequence reads, the researchers present smooth gene expression trajectories in absolute transcript numbers. During a developmental period approximating the first 8 weeks of human gestation, transcript kinetics vary by eight orders of magnitude. Ordering genes by expression dynamics, they found that “temporal synexpression” predicts common gene function. Remarkably, a single parameter, the characteristic timescale, can classify transcript kinetics globally and distinguish genes regulating development from those involved in cellular metabolism.

Temporal Synexpression in Clutch A Poly(A)+


(A) Temporal map of the transcriptome: an enumeration of all gene expression dynamics in the embryo. Heatmaps display all genes normalized by maximum expression and ordered by similarity. Inset (right) expands on genes transiently expressed with early onset. Vertical bars mark GO enrichment (colors correspond to GO terms), numbers appended to GO bars indicate the number of genes of given category. All reported GO blocks are enriched with p < 2 × 10−4 (Fisher’s exact test) for entire block. S1, S2, V1, and V2 mark the locations of somite and vision temporal synexpression genes, respectively, in (B). “Transcription Factors” labels an annotation of transcription factors separate to GO. (B) Somite (left) and vision (right) synexpression groups.

Overall, this analysis provides unprecedented insight into the reorganization of maternal and embryonic transcripts and redefines our ability to perform quantitative biology.

Owens ND, Blitz IL, Lane MA, Patrushev I, Overton JD, Gilchrist MJ, Cho KW, Khokha MK. (2016) Measuring Absolute RNA Copy Numbers at High Temporal Resolution Reveals Transcriptome Kinetics in Development. Cell Rep 14(3):632-47. [article]

Leave a Reply

Your email address will not be published. Required fields are marked *


Time limit is exhausted. Please reload CAPTCHA.