The expression of Lin28B, let-7b, and let-7g in ovarian granulosa cells 24, 48, and 72 h
after transfection was assessed using qRT-PCR.
For Lin28B mRNA expression, ACTB was selected as the reference gene for normalizing
mRNA levels. The primer sequences for Lin28B and ACTB mRNA are presented in Table 1.
For let-7b and let-7g quantification, cDNA was synthesized using a miRNA RT Kit (Takara,
Dalian, China). The mature sequences of let-7b and let-7g were obtained using miRBase
(https://www.mirbase.org/ , last access: 18 April 2022) and were used to design the primers. The forward
primers for let-7b and let-7g were designed, whereas the reverse primers were included in
the Takara miRNA RT Kit. U6 was selected as a reference gene. Table 1 presents
the primer sequences used for let-7b, let-7g, and U6 miRNA.
The qRT-PCR was performed using the Mir-X miRNA qRT-PCR TB Green Kit (Takara,
Dalian, China). All experiments were run on an Eppendorf device (Eppendorf,
Germany). Each sample and assay were run in triplicate. The levels of Lin28B,
let-7b, and let-7g were calculated using the 2 method.
after transfection was assessed using qRT-PCR.
For Lin28B mRNA expression, ACTB was selected as the reference gene for normalizing
mRNA levels. The primer sequences for Lin28B and ACTB mRNA are presented in Table 1.
For let-7b and let-7g quantification, cDNA was synthesized using a miRNA RT Kit (Takara,
Dalian, China). The mature sequences of let-7b and let-7g were obtained using miRBase
(
primers for let-7b and let-7g were designed, whereas the reverse primers were included in
the Takara miRNA RT Kit. U6 was selected as a reference gene. Table 1 presents
the primer sequences used for let-7b, let-7g, and U6 miRNA.
The qRT-PCR was performed using the Mir-X miRNA qRT-PCR TB Green Kit (Takara,
Dalian, China). All experiments were run on an Eppendorf device (Eppendorf,
Germany). Each sample and assay were run in triplicate. The levels of Lin28B,
let-7b, and let-7g were calculated using the 2 method.