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Relationship of quantitative reverse transcription polymerase chain reaction (RT-PCR) to RNA Sequencing (RNAseq) transcriptome identifies mouse preimplantation embryo reference genes†.
Tscherner, Allison K; McClatchie, Taylor; Macaulay, Angus D; Baltz, Jay M.
Afiliação
  • Tscherner AK; Ottawa Hospital Research Institute, Ottawa, ON, Canada.
  • McClatchie T; Department of Obstetrics and Gynecology, University of Ottawa Faculty of Medicine, Ottawa, ON, Canada.
  • Macaulay AD; Ottawa Hospital Research Institute, Ottawa, ON, Canada.
  • Baltz JM; Department of Obstetrics and Gynecology, University of Ottawa Faculty of Medicine, Ottawa, ON, Canada.
Biol Reprod ; 109(5): 601-617, 2023 11 15.
Article em En | MEDLINE | ID: mdl-37669129
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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Transcrição Reversa / Transcriptoma Limite: Animals Idioma: En Revista: Biol Reprod Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Canadá

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Transcrição Reversa / Transcriptoma Limite: Animals Idioma: En Revista: Biol Reprod Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Canadá