Whole transcriptome amplification

We collected oocytes 8 hours (just before insemination) after injection with miR-320 inhibitor (n = 80) or NC inhibitor (n = 50) and measured the expression levels of 13 genes (Apc, Aspm, Btrc, Csnk1a1, Ctnna1, Ctnnb1, Dvl3, Gsk3b, Lef1, Lrp6, Numb, Tcf7 and Wnt7a) within or regulate Wnt signaling pathway by qRT-PCR. Prior to the PCR, whole transcriptome amplification (TaKaRa, Dalian, China) was performed due to the limited quantity of RNA in the small numbers of oocytes. Experiments were performed in triplicate.

We collected the human and mouse oocytes 8 h (just before insemination) after injection with the miR-451 inhibitor (human oocytes: n = 21; mouse oocytes: n = 160) or the NC inhibitor (human oocytes: n = 20; mouse oocytes: n = 95). The miRNeasy Micro Kit (QIAGEN) was used for isolation and purification of RNA from oocytes according to the manufacturer’s protocol [14 (link)]. The expression levels of 12 target genes (WNT4, AXIN1, COX2, CDX2, CTNNB1, WNT5A, WNT3, WNT8B, CCND1, c-MYC, ATP2, and MMP9) within or regulating the WNT signalling pathway were measured by qRT-PCR in human/mouse oocytes and in 2-cell and blastocyst-stage embryos. The measurements were then compared between the miR-451 inhibitor-injected and control groups. Prior to PCR, whole transcriptome amplification (TaKaRa, Dalian, China) was performed because the quantity of RNA was limited due to the small number of oocytes. qRT-PCR reactions were performed in triplicate for each sample.