Abstract
With the progress in our understanding of germ cell development, there is an emerging need to investigate the mechanisms of mRNA translation functioning in these cells. Indeed, posttranscriptional regulations of gene expression drive the most important transitions of the germ cell life cycle. Here we describe a strategy to measure mRNA translation in the oocyte, taking advantage of an approach originally developed to identify the transcriptome of a subgroup of cells in a complex cell mixture. This technique takes advantage of the “RiboTag” approach to express an HA-tag on the large ribosomal subunit of the ribosomes in the oocyte. Immunoprecipitation of the extracts followed by qPCR or RNAseq is used to identify mRNAs actively translated.
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Notes
- 1.
The volume of slurry will vary according to the number of samples planned. It is important to remember that each sample needs to be divided into 2 (HA antibody and control IgG).
- 2.
Always add an excess of 20–30 μL of magnetic beads to preclear the sample and another 20–30 μL to compensate for pipetting errors.
- 3.
The number of oocytes can vary; in our past experience in the lab, we were able to collect sufficient RNA with a minimum of 75 oocytes per RNA-IP (HA antibody and IgG).
- 4.
This volume was determined because we use all the RNA for the cDNA synthesis reaction using the Reverse-Transcription SupIII Kit from Invitrogen, which uses 8 μL of RNA per reaction.
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Acknowledgments
We are grateful to Dr. Stanley McKnight for assistance during the setup of the technique. We are also grateful to Mr. Bruno Felício for his support with the figure preparation. The work done in the authors’ laboratory is supported by NIH grants P50 HD055764 R01 GM115241 and R01 GM116926
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Martins, J.P.S., Conti, M. (2018). Profiling Maternal mRNA Translation During Oocyte Development. In: Verlhac, MH., Terret, ME. (eds) Mouse Oocyte Development. Methods in Molecular Biology, vol 1818. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8603-3_6
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DOI: https://doi.org/10.1007/978-1-4939-8603-3_6
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