Rt kit

Total RNA was extracted from the cell lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized using an RT kit (Toyobo Life Science, Osaka, Japan) according to the manufacturer's protocol. Dicer and β-actin were measured by a SYBR Green (Takara Bio, Inc., Otsu, Japan) qPCR assay. The sequences of the Dicer primers were as follows: Upstream, 5′-GTGGTTCGTTTTGATTTGCCC-3′ and downstream, 5′-CGTGTTGATTGTGACTCGTGGA-3′ (NM_001195573.1). β-actin was used for normalization, and the primer sequences were as follows: Upstream, 5′-GCCAACACAGTGCTGTCTGG-3′ and downstream, 5′-GCTCAGGAGGAGCAATGATCTTG-3′. Dicer was amplified under the following qPCR reaction conditions: Initial denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 sec, and annealing at 62°C for 1 min. All reactions were run on an Applied Biosystems 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2^−ΔΔCq method was used to calculate relative changes in gene expression (13 (link)).

Total RNA was extracted from frozen tissues or cells using TRIzol^® reagent (Life Technologies; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was transcribed to first-strand cDNA using an RT kit (Toyobo Life Science, Osaka, Japan). The RT-qPCR protocol was performed using SYBR-Green PCR master mix (Bio-Rad Laboratories, Hercules, CA, USA) and an Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific, Inc.). The primer sequences were: miR-33b forward, 5′-ATTCTTTCGAACTGTCTTGG-3′ and reverse, 5′-TCACCCTCGGCTGTCCTGACA-3′; U6 forward, 5′-AGTACCAGTCTGTTGCTGG-3′ and reverse, 5′-TAATAGACCCGGATGTCTGGT-3′. The qPCR cycle was set to an initial 95°C for 10 min, followed by 40 cycles of 95°C for 25 sec, 60°C for 30 sec, and 72°C for 30 sec. Gene expression levels were measured using the 2^−∆∆Cq method (13 (link)).

Total RNA was isolated using RNAiso (Takara, Dalian, China). The RT Kit (Toyobo Co. Ltd, Osaka, Japan) and KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) were used to measure the expression levels of LINC00336 using the Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific, Inc.) and normalized to the internal control U6. LncRNAs expression levels were calculated using the 2^–ΔΔCq method [21 (link)].

Total RNA was extracted from prostate tissue using TRIzol reagent (Sigma, MO, USA) and the absorbance of RNA samples were measured at 260 and 280 nm. Equal amounts of total RNA (1.4 µg) from the prostate tissue of each rat were subjected to reverse transcription (RT) using a commercially available RT kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed using SYBR Green Master Mix (Thermo Scientific, MA, USA). The following primer sequences were used: Il6, 5ʹ-TAGTCCTTCCTACCCCAACT −3ʹ (forward) and 5ʹ-TTGGTCCTTAGCCACTCCTT-3ʹ (reverse); Il8, 5ʹ-CATTAATATTTAACGATGTGGATGCGTTTCA-3ʹ (forward) and 5ʹ-GCCTACCATCTTTAAACTGCACAAT-3ʹ (reverse); Gapdh, 5ʹ-ACAGCAACAGGGTGGTGGAC-3ʹ (forward) and 5ʹ-TTTGAGGGTGCAGCGAACTT-3ʹ (reverse). Relative expressions are presented as fold-change in the cDNA level of the target gene relative to that of the endogenous control (GAPDH), determined using the 2^−ΔΔCt method, as described previously.16 (link)