Epigenetic-related transcriptional reprogramming elucidated by identification and validation of a novel reference gene combination for RT-qPCR studies in porcine oocytes of contrasting quality.
Mol Biol Rep
; 51(1): 368, 2024 Feb 27.
Article
in En
| MEDLINE
| ID: mdl-38411699
ABSTRACT
BACKGROUND:
Reliable RT-qPCR results are dependent on appropriate normalisation. Oocyte maturation studies can be challenging in this respect, as the stage of development can distinctively affect reference gene transcript abundance. The aim of this study was to validate the use of reference genes in oocyte in vitro maturation RT-qPCR studies, and thereafter, examine the abundance of transcripts supporting histone modification during oocyte and early embryo development in oocytes of contrasting quality. METHODS ANDRESULTS:
Total RNA from oocytes from prepubertal gilts and sows was extracted either directly succeeding follicle aspiration or after 44 h in vitro maturation, followed by RT-qPCR. The stability of YWHAG, HPRT1, ACTB, GAPDH, HMBS and PFKP, was analysed by NormFinder and further cross-validated by assessing results generated following application of different combinations of potential reference genes for normalisation of the RT-qPCR data. Combining ACTB and PFKP generated high stability according to NormFinder and concordant results. Applying this normalisation, gilt derived oocytes displayed significantly higher abundance than oocytes from sows of almost all the epigenetic-related transcripts studied (HDAC2, SIRT1, SALL4, KDM1A, KDM1B, KDM5A), both before and after maturation.CONCLUSIONS:
This study identified the combined use of ACTB and PFKP as the optimal normalisation for porcine oocyte RT-qPCR data. In oocytes collected from prepubertal gilts, transcription did not appear to be silenced at the time of aspiration, and accumulation of transcripts supporting histone modification facilitating proper fertilization and further embryo development seemed delayed. The results imply the epigenetic-related transcripts may have potential as markers of oocyte quality.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Oocytes
/
Sus scrofa
Limits:
Animals
Language:
En
Journal:
Mol Biol Rep
Year:
2024
Document type:
Article
Affiliation country:
Norway