Relative changes in the RNA contents of oocytes and preimplantation embryos of the mouse

Moore, G. P. M.; Lintern‐Moore, Sue; Scott-Murphy, Lisa

doi:10.1051/rnd:19810402

Cited by 5 publications

(4 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is particularly important for preimplantation embryo RT-qPCR datasets as it avoids the technical uncertainty associated with handling such limited amounts of total RNA. Furthermore, the sharp rise in an embryo’s total RNA content after the 8-cell stage [ 45 , 46 ] renders the use of alternative normalization bases, such as embryo count and exogenous spike-ins, inadequate. These issues are further exacerbated when analyzing the spent culture media.…”

Section: Discussionmentioning

confidence: 99%

Selecting Normalizers for MicroRNA RT-qPCR Expression Analysis in Murine Preimplantation Embryos and the Associated Conditioned Culture Media

Hawke

Watson

Betts

2023

JDB

View full text Add to dashboard Cite

Normalizing RT-qPCR miRNA datasets that encompass numerous preimplantation embryo stages requires the identification of miRNAs that may be used as stable reference genes. A need has also arisen for the normalization of the accompanying conditioned culture media as extracellular miRNAs may serve as biomarkers of embryo developmental competence. Here, we evaluate the stability of six commonly used miRNA normalization candidates, as well as small nuclear U6, using five different means of evaluation (BestKeeper, NormFinder, geNorm, the comparative Delta Ct method and RefFinder comprehensive analysis) to assess their stability throughout murine preimplantation embryo development from the oocyte to the late blastocyst stages, both in whole embryos and the associated conditioned culture media. In descending order of effectiveness, miR-16, miR-191 and miR-106 were identified as the most stable individual reference miRNAs for developing whole CD1 murine preimplantation embryos, while miR-16, miR-106 and miR-103 were ideal for the conditioned culture media. Notably, the widely used U6 reference was among the least appropriate for normalizing both whole embryo and conditioned media miRNA datasets. Incorporating multiple reference miRNAs into the normalization basis via a geometric mean was deemed beneficial, and combinations of each set of stable miRNAs are further recommended, pending validation on a per experiment basis.

show abstract

Section: Discussionmentioning

confidence: 99%

Selecting Normalizers for MicroRNA RT-qPCR Expression Analysis in Murine Preimplantation Embryos and the Associated Conditioned Culture Media

Hawke

Watson

Betts

2023

JDB

View full text Add to dashboard Cite

show abstract

“…However, total RNA and poly(A) mRNA contents have been determined by classical chemical and spectroscopic methods. Total RNA was determined by optical density measurements of unmodified [44], dye (Azur B)-reacted [24], or radiolabeled [45] RNA, which indicated that total RNA dropped from the MII egg to about 60–70% in 2-cell to 4-cell embryos before increasing to very high levels at the blastocyst stage (∼2.5–7-fold relative to GV oocytes, depending on blastocyst stage). Poly(A) RNA, which represents a portion of total mRNA, decreases steadily from the GV stage to the 2-cell stage which has a level of poly(A) RNA ∼27% that of the GV stage.…”

Section: Discussionmentioning

confidence: 99%

“…However, as discussed above, the total amount of mRNA in oocytes and preimplantation embryos varies considerably because of maternal mRNA degradation and stage-specific embryonic gene expression. For example, the total amount of mRNA in mouse embryos falls to a minimum at the 2-cell stage when maternal transcripts have been degraded but embryonic genome expression is just being initiated [24]. For this reason, a transcript that is found to be constant through the 2-cell stage by RNAseq (i.e., it is present at a similar fraction of the entire mRNA pool as at other stages) will necessarily be lower than at other stages when measured by RT-qPCR since it is a fraction of a smaller total pool of transcripts.…”

Section: Introductionmentioning

confidence: 99%

Relationship of quantitative reverse transcription polymerase chain reaction (RT-PCR) to RNA Sequencing (RNAseq) transcriptome identifies mouse preimplantation embryo reference genes

Tscherner,

McClatchie,

Macaulay

et al. 2023

Biology of Reproduction

View full text Add to dashboard Cite

Numerous reference genes for use with quantitative reverse transcription polymerase chain reaction (RT-qPCR) have been used for oocytes, eggs, and preimplantation embryos. However, none are actually suitable because of their large variations in expression between developmental stages. To address this, we produced a standardized and merged RNA sequencing (RNAseq) data set by combining multiple publicly available RNAseq data sets that spanned mouse GV oocytes, MII eggs, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos to identify transcripts with essentially constant expression across all stages. Their expression was then measured using RT-qPCR, with which they did not exhibit constant expression but instead revealed a fixed quantitative relationship between measurements by the two techniques. From this, the relative amounts of total messenger RNA at each stage from the GV oocyte through blastocyst stages were calculated. The quantitative relationship between measurements by RNAseq and RT-qPCR was then used to find genes predicted to have constant expression across stages in RT-qPCR. Candidates were assessed by RT-qPCR to confirm constant expression, identifying Hmgb3 and Rb1cc1 or the geometric mean of those plus either Taf1d or Cd320 as suitable reference genes. This work not only identified transcripts with constant expression from mouse GV oocytes to blastocysts, but also determined a general quantitative relationship between expression measured by RNAseq and RT-qPCR across stages that revealed the relative levels of total mRNA at each stage. The standardized and merged RNA data set should also prove useful in determining transcript expression in mouse oocytes, eggs, and embryos.

show abstract

“…These studies capitalized on the emergence of the first in vitro embryo culture systems (e.g., Blandau & Rumery, 1957; Brinster, 1963; Daniel, 1965; Daniel & Krishnan, 1967; Kane & Foote, 1970; Van Blerkom & Manes, 1974; Whitten & Biggers, 1968). Studies progressed soon thereafter to discover the major macro‐features of gene regulation, including temporal changes in protein, DNA, and RNA content and rates of synthesis as embryos progress through cleavage and cavitation (e.g., Chapman et al, 1971; Karp et al, 1974; Manes, 1969; Manes & Daniel, 1969; Mohla et al, 1970; Moore et al, 1981; Olds et al, 1973; Sternlicht & Schultz, 1981; Weitlauf, 1971). Major features of maternal messenger RNA (mRNA) and protein accumulation and mRNA stability in oocytes were discovered (Bachvarova, 1992; De Leon et al, 1983; Epstein & Smith, 1974; Paynton et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

Preimplantation embryo gene expression: 56 years of discovery, and counting

Latham

2023

Molecular Reproduction Devel

View full text Add to dashboard Cite

The biology of preimplantation embryo gene expression began 56 years ago with studies of the effects of protein synthesis inhibition and discovery of changes in embryo metabolism and related enzyme activities. The field accelerated rapidly with the emergence of embryo culture systems and progressively evolving methodologies that have allowed early questions to be re-addressed in new ways and in greater detail, leading to deeper understanding and progressively more targeted studies to discover ever more fine details. The advent of technologies for assisted reproduction, preimplantation genetic testing, stem cell manipulations, artificial gametes, and genetic manipulation, particularly in experimental animal models and livestock species, has further elevated the desire to understand preimplantation development in greater detail. The questions that drove enquiry from the earliest years of the field remain drivers of enquiry today. Our understanding of the crucial roles of oocyteexpressed RNA and proteins in early embryos, temporal patterns of embryonic gene expression, and mechanisms controlling embryonic gene expression has increased exponentially over the past five and a half decades as new analytical methods emerged. This review combines early and recent discoveries on gene regulation and expression in mature oocytes and preimplantation stage embryos to provide a comprehensive understanding of preimplantation embryo biology and to anticipate exciting future advances that will build upon and extend what has been discovered so far.

show abstract

Relative changes in the RNA contents of oocytes and preimplantation embryos of the mouse

Cited by 5 publications

References 30 publications

Selecting Normalizers for MicroRNA RT-qPCR Expression Analysis in Murine Preimplantation Embryos and the Associated Conditioned Culture Media

Selecting Normalizers for MicroRNA RT-qPCR Expression Analysis in Murine Preimplantation Embryos and the Associated Conditioned Culture Media

Relationship of quantitative reverse transcription polymerase chain reaction (RT-PCR) to RNA Sequencing (RNAseq) transcriptome identifies mouse preimplantation embryo reference genes

Preimplantation embryo gene expression: 56 years of discovery, and counting

Contact Info

Product

Resources

About