Total RNA extraction and RT-qPCR

For RT-qPCR, 12 rats were randomly classified into 2 groups (6 rats/group): The control (saline) and model (6-OHDA). Carbon dioxide asphyxiation was performed to sacrifice the animals. A total of 11 rats (one rat from the model group that did not reach the modeling standard was excluded from the experiment) were placed in a transparent and airtight chamber (40×20×20 cm) and gassed with carbon dioxide at a rate of 30% of the chamber volume per minute. The tissues were collected after the animal death was ensured by observing the movement, heartbeat and respiration. DNA-free RNA was obtained from SN tissue or PC12 cells using the Total RNA Extraction kit (cat. no. LS1040; Promega Corporation), and 500 ng of total RNA were reverse transcribed using the PrimeScript RT reagent kit (cat. no. RR600A, Takara Bio, Inc.) according to the manufacturer's protocol, at 37°C for 15 mins and 85°C for 30 sec. qPCR was performed in triplicate with TB Green Premix Ex Taq II (cat. no. RR820A, Takara Bio, Inc.) using a Light Cycler 480 SYBR-Green I Master Mix (Roche Diagnostics, GmbH). The amplification process was as follows: Denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 30 sec and extension at 50°C for 30 sec. The U6 gene was used for the normalization of miRNA expression. The primers for qPCR were as follows: miR-335 RT primer, 5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG ATA CGA CAC ATT T-3′; forward, 5′-CGG CGC TCA AGA GCA ATA ACG AA-3′ and reverse, 5′-ATC CAG TGC AGG GTC CGA GG-3′; U6 RT primer 5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA AA A TA-3′; forward, 5′ AGA GAA GAT TAG CAT GGC CCC TG-3′ and reverse, 5′-ATC CAG TGC AGG GTC CGA GG-3′. Relative expression levels were analyzed using the 2^−ΔΔCq method (36).