Abstract
Changes in abundance of mRNAs during oocyte growth and maturation and during pre-implantation embryo development have been documented using quantitative real-time RT-PCR (qPCR), microarray analyses, and whole genome sequencing. However, these techniques require amplification of mRNAs, normalization using housekeeping genes, can be biased for abundant transcripts, and/or require large numbers of oocytes and embryos which can be difficult to acquire from mammalian species. We optimized a single molecule RNA fluorescence in situ hybridization (RNA-FISH) protocol, which amplifies fluorescence signal to detect candidate transcripts, for use with individual oocytes and embryos. Quantification using the software Localize showed patterns of Gdf9 and Pou5f1 mRNA expression in oocytes and embryos that were consistent with previously published data. Interestingly, low levels of Nanog mRNA were also accurately and reproducibly measured in oocytes and one- and two-cell embryos suggesting that RNA-FISH could be used to detect and quantify low abundance transcripts. Unlike other techniques, RNA-FISH is also able to detect changes in the localization patterns of mRNAs which may be used to monitor post-transcriptional regulation of a transcript. Thus, RNA-FISH represents an important technique to investigate potential mechanisms associated with the synthesis and stability of candidate mRNAs in mammalian oocytes and embryos.
Highlights
It is undeniable that these collective methodologies have produced a wealth of information about relative changes in the abundance of mRNAs during important periods of oocyte maturation and embryonic development
A proprietary negative control probe which recognizes the Bacillus subtilis dihydrodipicolinate reductase (DapB) mRNA was purchased (Table 1). Each of these probes were designed based on published NCBI sequence data for the murine or B. subtilis RNAs, respectively using an algorithm described by Bushnell et al.[23] to optimize specificity
In situ hybridization (ISH) for detection of DNA and RNA in histological sections was described by Pardue and Gall[29,30] and John et al.[31] in 1969
Summary
It is undeniable that these collective methodologies have produced a wealth of information about relative changes in the abundance of mRNAs during important periods of oocyte maturation and embryonic development. Most of the data represents the average relative abundance of a transcript in a pool of cells that has often times been normalized to a constitutively expressed housekeeping transcript. Assays cannot identify changes in mRNA localization, which is an important component of post-transcriptional regulation of RNA storage, translation, and degradation including in maturing oocytes and pre-implantation embryos[10,11,12,13]. To determine the accuracy and validity of this method in oocytes and embryos, we analyzed the absolute abundance and localization of three well described transcripts, (Gdf[9], Pou5f1, and Nanog) in mouse oocytes and embryos
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