Primescript rt master mix

Total RNA was extracted from the cells by total RNA extraction kit according to the manufacturer’s instructions. Additionally, the concentration and quality of total RNA was determined by Nano Drop. cDNAs for each RNA sample were prepared using PrimeScript RT Master Mix, then stored at −20 °C.
The mRNA expression levels of Bax, Bcl-2, caspase 3, caspase 8, and Cytc, were quantified using ABI-prism 7500 sequence detection system (Applied Biosystems, Inc., Foster City, CA, USA) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GADPH) reference gene. As shown in Table 1, the primers sequences were designed using Prime Premier 5.0 and synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. (Shanghai, China).
Real-time quantitative PCR reaction was carried out in 20 μL reaction mixtures, containing 10 μL of SYBR Premix Ex Taq (2×), 0.4 μL of ROX Reference Dye (50×), 0.8 μM of each forward and reverse primers, cDNA aliquots, and nuclease-free water. Real-time PCR amplification was performed with an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of 5 s at 95 °C and 31 s at 60~64 °C. The 2^−ΔΔCt method was used to determine the relative expression of each gene compared to a reference gene. All samples were amplified in a single PCR run, and each amplification was repeated at least three times [49 (link)].

Beclin 1, mTOR, and p53 are upstream and downstream regulatory signals of autophagy activity, and their levels can indirectly reflect the degree of autophagy activity [15 (link)–17 (link)]. CP-H203 and CAL-27 cells in control and chemotherapy groups cultured in 2.1 were selected for detection, and the levels of miR-214, ULK1, Beclin 1, mTOR, and p53 in the above cells were detected by PCR. TRIzol reagent isolated total RNA from cells following the instructions (Life Technologies, Thermo Fisher Scientific). qRT-PCRs of mRNAs were reverse-transcribed using the Prime Script RT Master Mix (Applied Biosystems, Foster City, CA), and reactions on the obtained cDNAs were conducted on the ABI StepOneTM Real-Time PCR System (Applied Biosystems, Foster City, CA). See Table 1 for primer information. Transcription alterations relative to β-actin or U6 were assessed using 2^−ΔΔCt.

Harvested root and leaf samples were snap-frozen in liquid nitrogen prior to storage at −80°C. Total RNA was extracted from the seeding roots with a commercial kit (Aidlab Biotechnologies Co., Ltd. Beijing, China), and RNA purity and concentration were analyzed using an ND−2000 spectrometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA integrity was analyzed with 1.5% agarose gel electrophoresis (180 V, 15 min). A Takara PrimeScript RT Master Mix was used for cDNA synthesis, and qPCR reactions were performed using a 7300 Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA, USA) with the primers listed in Table 1.

Total RNA was isolated from prostate tissue samples and cell lines with TRIzol (TaKaRa, China) following all manufacturer protocols. We treated total RNA with DNase I (TaKaRa, Dalian, China) to remove genomic DNA. Reverse transcription of purified RNA was carried out using random primer sets and standardized reaction conditions with the use of PrimeScript RT Master Mix (Applied Biosystems, Foster City, CA). Quantitative real-time PCR (qRT-PCR) was carried out on the ABI7300 system (Applied Biosystems) following all manufacturer protocols. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6 was used as internal controls. The 2^-ΔΔCt method was used to assess relative transcription alterations according to a previous study [14 (link)]. qPCR primer sequences are shown in Table 1.

Total RNA was extracted by using TRIzol reagent (Invitrogen, USA). The concentration of RNA was determined by a NanoDrop2000 spectrophotometer and converted into cDNA with PrimeScript RT Master Mix (Invitrogen, USA). Primers were designed with Primer5.0 software according to the gene sequence in the GenBank database and synthesized by Nanjing Qinke Co., Ltd. The reaction system for miRNA quantitative real-time polymerase chain reaction (qPCR) was designed according to the instructions of a One Step SYBR® PrimeScript® PLUS RT-PCR Kit (Takara, Japan). miRNA expression was normalized using the 2^−ΔΔCt method from the Ct values relative to the housekeeping gene U6. Primer sequences are listed in Supplementary Table 1.