Applied biosystems stepone

The fresh hippocampal tissues were put in commercial RNA extraction reagent (Roche Applied Science, Indianapolis, IN, USA) for quantitative polymerase chain reaction (qPCR) analysis. First-strand cDNA was synthesized with the use of 1 μg of total RNA (Transcriptor First Strand cDNA Synthesis Kit, Roche Applied Science). The qPCR was performed with Applied Biosystems Step One (Applied Biosystems, Foster City, CA, USA) using SYBR Green Master solution (Roche Applied Science) to measure the fluorescence intensity of amplified products. Reactions were as follows: 55°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. Data were analyzed using the 2^−ΔΔCt method, as previously described (Avnet et al., 2017), with β-actin as a housekeeping gene. Fold change of all groups was normalized to those of the control group. The sequences of primers are shown in Table 1. All the sequence specificities of the primers used in the current study have been verified by Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/).

The fresh hippocampal tissues were subjected to total RNA extraction using a commercially available assay (Roche) according to the manufacturer's protocol. First-strand cDNA was synthesized with the use of 1 μg of total RNA (Transcriptor First Strand cDNA Synthesis Kit, Roche). The qPCR was performed with Applied Biosystems StepOne (Applied Biosystems, Foster City, CA, USA) using SYBR Green Master (Roche) to measure the fluorescence intensity of amplified products. Reactions were as follows: 55°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s followed by 60°C for 1 min. Data were analyzed by the 2^−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene. Fold change of all groups was compared with that of 3-month-old WT group, which was set as one. The sequences of primers are shown in Table 1. All the sequence specificities of the primers used in the current study had been verified by Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/).

To detect EGFR gene expression level, cells were harvested after being treated with aX-Td@bsiRNA, free siRNA, and lipo3000@siRNA for 48 h. Total RNA was extracted from cells using RNeasy Mini Kit (QIAGEN, Germany), then reverse-transcribed with HiScript® Q Select RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China) to obtain cDNAs. Real-time PCR was performed using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus), ROX Plus (TAKARA, Dalian, China) and amplified with Applied Biosystems Stepone™ (Thermo Fisher Scientific, USA). All samples’ EGFR mRNA expression levels were normalized by β-actin amplification. Primer sequences used for EGFR (forward 5ʹ-AGACGCAGATAGTCGCCCAAAG-3ʹ, reverse 5ʹ-TCCATCAGGGCACGGTAGAAG-3ʹ) were designed and synthesized by TAKARA (Dalian, China). β-Actin (forward 5ʹ-AAATCGTGCGTGACATTAA-3ʹ, reverse 5ʹ-CTCGTCATACTCCTGCTTG-3ʹ)³⁸ (link) was synthesized by TAKARA (Dalian, China).

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from the clinical BPH, PCa and adjacent tissues. A NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.) was used to assess the purity and quantity of the isolated RNA. Complementary DNA (cDNA) was synthesized using the Applied Biosystems StepOne (Thermo Fisher Scientific, Inc.) with qPCR kit and SYBR-Green master mix (Roche Diagnostics.). According to the instructions of the RT kit (PrimeScript^™ RT Master Mix; Takara), RT was performed as follows: Initiation at 37˚C for 15 min, then 85˚C for 5 sec. The qPCR conditions included an initiation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 1 min. The 2^-ΔΔCq method (18 (link)) was performed to analyze relative quantities for the level of mRNA expression. The specific primer sequences were as follows: 5'-TGGCCTCAGCTAGGTAACCA-3' (forward) and 5'-GTACCTGGGAGCTGTCATCG-3' (reverse) for SPC24. 5'-GAAGCGCAGTTCAGTTTCC-3' (forward) and 5'-GGTTTCTCTTTGGTTTGAGGG-3' (reverse) for NDC80. 5'-TGGGAAAGATACATACAGTGG-3' (forward) and 5'-GAATCTTGGGTCATTGTGGT-3' (reverse) for BUB1.