Accupower rocketscript cycle rt premix

The amount of PSD-95 mRNA in SH-SY5Y cells was evaluated with quantitative real-time PCR. Total cellular RNA was extracted from SH-SY5Y cells using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) and complementary DNA (cDNA) was synthesized using AccuPower Rocketscript Cycle RT Premix (Bioneer, Daejeon, Korea). The cDNA was mixed with SYBR premix EX-Taq RT-PCR Kit (Takara, Shiga, Japan) and specific primers in a total reaction volume of 20 μL. PCR was performed using the following primers: PSD-95 forward: 5′-TCGGTGACGACCCATCCAT-3′, PSD-95 reverse: 5′-GCACGTCCACTTCATTTACAAAC-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 5′-ACAACTTTGGTATCGTGGAAGG-3′, GAPDH reverse: 5′-GCCATCACGCCACAGTTTC-3′. GAPDH was used as an internal control. The ΔCt values from Aβ₄₂-treated SH-SY5Y cells were compared with those from untreated SH-SY5Y cells.

The organoids (Passage 4) were treated with Cell Recovery Solution (Corning Incorporated, Corning, NY, USA) to remove the Matrigel, and total RNA was extracted using the MagListo™ 5 M Cell Total RNA Extraction Kit (Bioneer, Daejeon, Republic of Korea) according to the manufacturer's guidelines. Subsequently, the RNA product was subjected to a quantitative reverse transcription-polymerase chain reaction (RT-PCR). Firstly, RNA was synthesized to complementary DNA (cDNA) using AccuPower® RocketScript™ Cycle RT Premix (Bioneer). Quantitative polymerase chain reaction was performed by mixing the cDNA with AccuPower® 2X Greenstar qPCR Master Mix (Bioneer). The mRNA expression level was measured with Thermal Cycler Dice® Real Time System III (Takara, Shiga, Japan). The primer sequences are given in Table 2. The negative control (no cDNA template) was ran simultaneously with every assay, and PCR from each cDNA sample was ran in triplicate. Constitutively expressed GAPDH was used as an endogenous control to correct any potential variations in RNA loading or reaction efficiency. The results were presented as relative fold changes using the GAPDH reference and applying the formula 2^-ΔΔCt. The PCR products were loaded into 1% agarose gel and visualized with a DNA gel staining solution under UV light.

Quantitative real-time PCR was performed for CSF-1, E-cadherin, and 18S (as a reference gene) mRNAs. MDA-MB-231 cells (1.2 × 10⁵ cells/well) were seeded into a 12-well plate and incubated at 37 °C with 5% CO₂ for 24 h. These cells were treated with indicated compounds (YL2, adamantane, ND1-YL2, or YL2-HyT6) for 18 h in Opti-MEM medium. After cells were washed with cold DPBS, mRNAs were isolated from these cells using an AccuPrep Universal RNA extraction kit (Bioneer, Daejeon, Korea). Then 1.5 µg of mRNAs were reverse-transcribed using AccuPower RocketScript Cycle RT PreMix (Bioneer) to generate complementary DNAs. RT-qPCR for CSF-1, E-cadherin, and 18S was performed with a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) and SYBR Green mix (Applied Biosystems) according to the manufacturer’s instructions with gene-specific primers (Table 1).

Total RNA was isolated from BV2 cells using TRIZOL reagent (Invitrogen Co, Grand Island, NT, USA) according to the manufacturer’s instruction. cDNA was synthesized by reverse transcription from 1 μg of total RNA using AccuPower Rocketscript cycle RT premix (Bioneer, Daejeon, Korea). Aliquots of cDNA were used for PCR using primer sets specific to iNOS, COX-2, IL-6, TNF-α, and GAPDH as a control. Used primers are as follows: iNOS sense: 5′-AGACCTCAACAGAGCCCTCA-3′, antisense: 5′-GCAGCCTCTTGTCTTTGACC-3′; COX-2 sense: 5′-GGAGAGACTATCAAGATAGT-3′, antisense: 5′-ATGGTCAGTAGACTTTTACA-3′; IL-6 sense: 5′-CCGGAGAGGAGACTTCACAG-3′, antisense: 5′-TCCACGATTTCCCAG-AGAAC-3′; TNF-α sense: 5′-TCAGCCTCTTCTCATTCCTG-3′, antisense: 5′-TGAAGAGAACCTGGGAGTAG-3′; GAPDH sense: 5′-TTGCAGTGGCAAAGTGGAGA-3′, antisense: 5′-CGTGGTTCACACCCATCACAA-3′.