The RNAeasy Animal RNA Isolation kit (Beyotime, China) was used to extract total RNA. cDNA was converted from mature miRNAs through the application of Hifair cDNA Synthesis SuperMix kit (Yeasen, China). To examine the expression of miR-519d, miR-328-3p and Ki-67, qRT-PCR was executed on QuantStudio 7 Flex (ThermoFisher, USA) using the BeyoFast SYBR Green qPCR Mix kit (Beyotime, China). The primers are summarized in Table 1 . Quantification of RNA levels was normalized with U6 or GAPDH using the 2−ΔΔCt cycle threshold method.
Primers Used for qRT-PCR
| Primers | Primer Sequences |
|---|---|
| miR-519d | Forward 5ʹ-TGCGGCAAAGTGCCTCCCTTTAG-3’ |
| Reverse 5ʹ-TGCGGGTGCTCGCTTCGGCAGC-3’ | |
| miR-328-3p | Forward 5ʹ-CGGGCCTGGCCCTCTCTGCC-3’ |
| Reverse 5ʹ-CAGCCACAAAAGAGCACAAT-3’ | |
| U6 | Forward 5′-GCTTCGGCAGCACATATACTAAAAT-3′ |
| Reverse 5′-CGCTTCACGAATTTGCGTGTCAT-3′ | |
| Ki-67 | Forward 5′-GGCACTTTGGATGCTCTCAGGC-3′ |
| Reverse 5′-GCATTTATGCCTGAGAGCATCC-3′ | |
| GAPDH | Forward 5′-GTCCATGCCATCACTGCCACCCAG-3′ |
| Reverse 5′-CTGATACATTGGGGGTGGGGACAC-3′ |