Sybr primescript plus rt pcr kit

The RNAiso Plus (Takara Bio, 9108) was used to extract total RNA and the subsequential reverse transcription was conducted with a PrimeScript™ RT reagent Kit (Takara Bio, RR047A). The resulting cDNAs or DNAs were quantified using real-time PCR with a SYBR PrimeScript PLUS RT-PCR Kit (Takara Bio, RR096A) on the Agilent Mx3000P Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA), using the following primers: BNIP3 forward primer, 5′-ACCACAAGATACCAACAGAG-3′; BNIP3 reverse primer, 5′-AGAGCAGCAGAGATGGAA-3′; β-actin forward primer, 5′-CTGTGCTATGTTGCCCTGGACTTC-3′; β-actin reverse primer, 5′-GCTCGTTGCCGATGGTGATGAC-3′. The relative expression was calculated using the 2-ΔΔCT method.

Total RNA was extracted from prostate tissue or LNCaP cells using Trizol (Takara, Japan). For miRNAs qRT-PCR, a total of 10 ng of RNA was transcribed into cDNA using the Taqman miRNA reverse transcription kit (Takara, Japan). And real-time PCR was performed with specific sense primers (Supplementary Table S1) and general antisense primer supplied by miRNA PrimeScript RT Enzyme Mix Kit (Takara, Japan) according to the manufacturer’s instructions. Quantitative c-myc gene-expression analysis was performed with specific primers (Supplementary Table S2) by using one step SYBR PrimeScript plus RT-PCR kit (Takara, Japan) according to the manufacturer’s instructions. MiRNAs and mRNA expression were normalized to RNU6 and GAPDH, respectively, using the 2^−∆∆Ct method.

The rats were sacrificed on schedule, and the retinal tissue was peeled from the eyes and rapidly frozen in liquid nitrogen. Total RNA was isolated from the tissue by using TRIzol reagent (Takara, Shiga, Japan) and incubated with chloroform for 15 min. After 15 min of centrifugation at 12,000× g 4°C, isopropanol was added to it and the admixture underwent a centrifugation at 12,000× g at 4°C for 15 min. Then, 75% ethanol prepared with RNase-free water was used to wash the RNA, and diethyl pyrocarbonate water resuspended the RNA. Afterward, cDNA was synthesized with PrimeScript RT reagent kit (Takara, Shiga, Japan) at 37°C for 15 min followed by 85°C for 5 s. qRT-PCR was performed by SYBR PrimeScript Plus RT-PCR Kit (Takara, Shiga, Japan) according to the operating instructions and applied with ABI 7500 PCR system (ABI, USA). The reaction was performed with 40 amplification cycles of denaturation at 95°C for 30 s followed by 94°C for 5 s and 60°C for 34 s. The quantity of target mRNA in test samples was detected and normalized to β-actin. The 2^−ΔΔct method was used to quantify the relative gene expression. Each sample was tested in triplicate. The Table 1 displays the sequences of the primers applied in this study.