Steponeplus real time rt pcr system

Total RNA was extracted from cultured bone marrow mesenchymal stem cells after 7 and 21 days, using TRIzol^® Reagent according to the manufacturer’s protocol, and reverse transcribed using TaqMan reverse transcriptase-PCR (Life Technologies, Carlsbad, CA, USA). ALP and RUNX2 were quantified at 7 days by real-time quantitative PCR on a Step One™ Plus Real-Time RT-PCR system (Applied Biosystems, Thermo Fisher Scientific, Tokyo, Japan), using the 2^−∆∆Ct method as previously described [52 (link)]. BMP was quantified in the same manner at 21 days.

rDNA transcription was determined by qPCR as previously described Bywater et al. [15 ]. Briefly, SK-UT-1 cells (1.0 × 10⁵) were seeded into 100 mm cell culture dishes and incubated for 12 h, following which the medium was exchanged with fresh complete medium containing vehicle or various concentrations of CX-5461, then incubated for 1 h. RNA was then extracted using a RNeasy^® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA (500 ng) was DNase treated (according to kit instructions), then incubated in T100™ Thermocycler (Bio-Rad, CA, USA) using the settings detailed in Supplementary Table 3. cDNA synthesis was performed using SuperScript™ IV Reverse Transcriptase (Invitrogen, Carlsbad, CA), Hexamer Random Primers (Promega, Madison, WI), and dNTPs (Invitrogen, Carlsbad, CA) with incubation in a T100™ Thermocycler (detailed in Supplementary Table 3). RT-PCR was performed using SYBR™ Green master mix (Applied Biosystems, Foster City, CA) and primers (Supplementary Table 4) in a StepOne™ Plus Real Time (RT) PCR System (Applied Biosystems) with the settings detailed in Supplementary Table 3.

Total cellular RNA was isolated from 3T3-L1 adipocytes using QIAzol lysis reagent (Qiagen Sciences, Maryland, USA). Total RNA was used as a template for first-strand cDNA synthesis performed using a Power cDNA Synthesis Kit (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer's instructions. The Real-Time RT-PCR mixture, with a final volume of 20 μL, consisted of Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μM of a forward primer, 1 μM of a reverse primer, and 0.1 μg of a cDNA sample. The thermal cycling conditions were as follows: holding stage, 10 s at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C, and then melt curve stage, 15 s at 95°C, 1 min at 60°C, and 15 s at 95°C. PCR products were measured with a StepOnePlus Real-Time RT-PCR System (Applied Biosystems, Foster City, CA, USA), and the relative gene expression was calculated based on the comparative CT method using a StepOne Software v2.1 (Applied Biosystems, Foster City, CA, USA). The mRNA expression of GAPDH was used as an endogenous control. The target cDNA was amplified using the sense and antisense primers described in S2 Table.

Real-time RT-PCR analysis was performed essentially as described previously [7] (link). Briefly, total RNA was extracted from cell lines and ovarian cancer tissues using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The mRNA levels of NIK, Cyclin D1 and MMP-9 were quantified using StepOnePlus real-time RT-PCR system (Applied Biosystems, Foster City, CA, USA). One hundred ng of total RNA was subjected to quantitative RT-PCR using TaqMan® One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems). Reverse transcription was performed at 48°C for 30 minutes and Taq DNA polymerase was activated at 95°C for 10 minutes, followed by 45 amplification cycles of denaturing at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute. The mRNA levels were normalized to the 18S ribosomal RNA levels measured by real-time RT-PCR using 100 pg of total RNA. Expression of miR-31 was examined using TaqMan® Micro RNA Assays (Applied Biosystems) according to manufacturer's instructions. The miR-31 levels were normalized with the RNU48 expression levels.

RD cells were seeded in six-well plates for 24 h. The virus (5 × 10⁶ PFU) was preincubated with DMSO or RA (280 μM) at 4°C for 1 h. The cells were then incubated with the virus for 1 h at 4°C to allow virus binding but not internalization. The cells were washed with PBS to remove unbound virus and then dissolved in the TRIzol reagent. Total RNA was extracted and 1 µg total RNA was reverse-transcribed using the M-MLV reverse transcriptase system (Invitrogen). Quantitative PCR (qPCR) was performed using the StepOnePlus real-time (RT) PCR system (Applied Biosystems, Foster City, CA) with the following specific primer pairs: VP2 forward primer 5′- CTGATGGCTTCGAATTGCAA-3′ and reverse primer 5′- GCGTTTATGTACGGCACTATTATTGT-3′; GAPDH forward primer 5′-TGCACCACCAACTGCTTAGC-3′ and reverse primer 5′-GGCATGGACTGTGGTCATGAG-3′. The target genes were then amplified under the following conditions: 50°C for 2 min, 95°C for 10 min, 50 cycles at 95°C for 15 s, and 60°C for 1 min. To quantify the changes in viral RNA expression, the 2^−ΔΔCT method was used to calculate relative fold changes normalized to the GAPDH control.

The expression of genes associated with osteogenic differentiation was evaluated by qRT-PCR. Total RNA was extracted from cells cultured for 3, 7, 14, and 21 days, and 1 µg was used to synthesize cDNA with the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA). ALP, Runx2, BMP-2, and OPN mRNA levels were evaluated by qRT-PCR on a StepOne Plus Real-Time RT-PCR system (Applied Biosystems). A 10 µL volume of Taqman Fast Universal PCR Master Mix, 1 µL of the 20× Taqman gene expression assay primer probe set, 2 µL of sample cDNA, and 7 µL of diethylpyrocarbonate-treated water (Nippongene, Toyama, Japan) were added to each well of a MicroAmp Fast Optical 96-well microplate (0.1-mL well volume; Applied Biosystems). The amplification reaction consisted of 40 cycles of 95 °C for 1 s and 60 °C for 20 s. Target gene expression levels were calculated relative to the negative control group with the 2^−ΔΔCt method.

Total RNA was extracted from BR, SR, or BR-SR mixture-treated cells using QIAzol lysis reagent (Qiagen Sciences Inc., Germantown, MD, USA) and GeneAll RiboEx Total RNA extraction kit (GeneAll Biotechnology, Seoul, Republic of Korea) as previously described [25 (link)]. mRNA evaluation was performed using a StepOnePlus Real-time RT-PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences used in this study are described in Table 2.