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Refined Deep Sequencing Technique Enables Profiling of Small RNAs in Mouse Embryos

NEW YORK (GenomeWeb) – Researchers from the Chinese Academy of Sciences have improved upon a deep sequencing technique so that it can be used to examine tiny amounts of RNA.

The academy's Ligang Wu and his colleagues tweaked the cDNA library construction method for small RNAs so that it only needs 10 nanograms of RNA, as they reported today in Science Advances. With this approach, they were then able to profile the small RNAs present in mouse oocytes and early embryos to examine their function during development.

"[Our] bioinformatics analyses show that the function of miRNA is suppressed from the oocyte to the two-cell stage and appears to be reactivated after the two-cell stage to regulate genes important in embryonic development," Wu and his colleagues wrote in their paper. "Our study thus provides a highly sensitive profiling method and valuable datasets for further examination of small RNAs in early embryos."

Typically, to profile small RNA through deep sequencing, hundreds of nanograms of total RNA are required. Low input, the researchers noted, leads to an abundance of ligation products between the 5' and 3' adaptors and thus affects the following rounds of PCR and usability of the sequencing reads.

But by optimizing the 5' and 3' adaptor ligation and PCR amplification steps, Wu and his colleagues lowered the amount of input RNA needed. In their tests using total RNA from 293 human embryonic kidney cell line cells, technical replicates of 100 nanograms, 33 nanograms, and 10 nanograms of total RNA all had highly similar microRNA profiles.

When they dropped the input RNA level below 10 nanograms, they observed reduced correlation coefficients between replications and decreased sensitivity in detecting more rare miRNAs.

"These results indicate that the performance of the improved small RNA sequencing method was robust and can be applied to other studies where RNA material is scarce," Wu and his colleagues wrote.

Using this library construction approach, the researchers then profiled the small RNAs present in oocytes, zygotes, and parthenogenetic one-cell, four-cell, and eight-cell mouse embryos. Each of these libraries, they noted, was created using fewer than 50 oocytes or embryos, and each of the technical replicates was highly correlated.

By comparing the small RNA profiles between the oocytes and zygotes, the researchers found that the first wave of miRNA production in the zygote starts as early as five hours after fertilization. Then as development progresses, various miRNAs are up- or downregulated, and many maternal-origin miRNAs are downregulated.

At the one- and two-cell stages, the researchers noted that zygotic miRNAs exhibited an increased amount of 3' mono- and oligoadenylation. They hypothesized that this may protect them from the degradation that occurs at that time.

Wu and his colleagues also found that a number of mRNAs predicted to be targeted by the top 20 most highly expressed, conserved miRNA families are degraded during the two-cell and four-cell stages. More lowly expressed miRNA families didn't experience such a decline. This suggested to the researchers that the repression activity of miRNA begins at the two-cell stage and increases gradually at subsequent stages.

The miRNA targets downregulated at this stage are involved in key signaling pathways, including Lin28, Stat3, and Nras.

"These results indicate that the regulatory function of miRNAs is reactivated to repress important stemness genes after the [two-cell] stage, thus promoting the development of embryos, an observation worth investigating in the future," Wu and his colleagues wrote.

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