Loading star

PCR amplifications were performed in a final reaction volume of 25 μl (1x buffer, 4.0 mM MgCl₂, 0.2 mM each dNTP) containing 1.25 U of GoTaq^® Flexi DNA polymerase (Promega, Madison, WI, USA), a 0.2 μM final concentration of each primer (Table 1) and 25 ng of template DNA. The amplification conditions included an initial denaturation period of 5 min at 95 °C, 35 cycles of denaturation (1 min at 95 °C), annealing (30 s at 62 °C), and extension (1 min at 72 °C), and a final extension period of 7 min at 72 °C. All conventional PCR reactions were performed using a PTC-225 thermocycler (MJ Research, Watertown, MA, USA). The amplified products were stained with LoadingStar (DYNEBIO, Seoul, Korea) and analysed by electrophoresis using 1.5% (w/v) agarose gels. The bands were visualised using a VersaDoc 1000 gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

Total RNA from the GA and the SOL muscles were isolated using the Trizol reagent (Takara, Otsu, Japan). Purity and concentration of RNA were determined spectrophotometrically using the NanoDrop Lite spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA was reverse-transcribed to cDNA with the RT premix at 42 ℃ for 60 min and then at 95 ℃ for 5 min using the SimpliAmp thermal cycler (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed with the synthesized cDNA, the SafeDry Taq PCR premix (CellSafe, Gyeonggi, Korea), and specific primers (Bioneer, Daejeon, Korea) (Table 1). Amplification steps comprised 34–36 cycles as follows: Denaturation for 30 s at 95 ℃, annealing for 30 s at 58–59 ℃, and extension for 45 s at 72 ℃. The final step included extension for 5 min at 72 ℃. After that, the amplified products stained with the Loading star (DyneBio, Daejeon, Korea) were separated on 1.5% agarose gel and detected with the G:BOX EF imaging system (Syngene, Cambridge, UK) and the Gene Snap program (Syngene). The Image J software (National Institutes of Health) was used for densitometry analysis. The β-actin served as the internal control.

To confirm the mRNA expression levels of various tumorigenic genes upon Pyri treatment, KBM5 cells were treated with 1 µM of Pyri. Cells were suspended with Trizol, then added chloroform, Iso-propanol to extract the RNA. Extracted RNA were reverse transcribed into cDNA, then reverse transcription polymerase chain reaction (RT-PCR) was performed. Each mRNA was performed with different temperature conditions: Bcl-2 reaction was performed at 94 °C for 15 s, 58 °C for 30 s, 72 °C for 1 min with 28 cycles and extension at 72 °C for 5 min. Bcl-xl reaction was performed at 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min with 30 cycles and extension at 72 °C for 7 min. Survivin reaction was performed at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 °C with 30 cycles and extension at 72 °C for 7 min. Cyclin D1 reaction was performed at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s with 35 cycles and extension at 72 °C for 10 min. All PCR products were separated on 1% agarose gel with Loading Star (Dynebio, Seongnam, Korea) and bands were detected by UV light. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control as described earlier [88 (link)].

Total RNA was extracted with a Trizol reagent, in accordance with the instructions from the manufacturer (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was changed to cDNA via reverse transcriptase, then amplified with a Taq polymerase via the use of an RT-PCR kit (Takara Bio Inc., Tokyo, Japan). The relative expressions of Bcl-xl, Bcl-2, Cyclin D1, Survivin, MMP-9, COX-2, Bax, and p21 were analyzed using a TaKaRa PCR Thermal Cycler (Code TP350, Takara Bio Inc., Tokyo, Japan) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The reaction was initially run for 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 55–60 °C, and 1 min at 72 °C, with a final period of 10 min at 72 °C. PCR products were run on 1% agarose gel, after which they were stained with loading star (Dynebio, Gyeonggi, Korea). The stained bands were visualized under UV light and then photographed.

The genotypes of the Drosophila lines were detected by PCR using EF Taq Polymerase (Solgent) following the manufacturer’s suggestion, in which 5 µL EF Taq buffer, 1 µL dNTPs, 0.5 µL forward primer (10 pmol/µL), and 0.5 µL reverse primer (10 pmol/µL) together with 50 ng genomic DNA in ddH₂O water to 50 µL made up the PCR mix. Drosophila genomic DNA was isolated using DNAzol (Invitrogen). After the initial denaturation at 95 °C for 5 min, PCR was carried out by denaturation at 95 °C for 30 sec, annealing at 57 °C for genotyping α-Syn, 65 °C for genotyping hOmi for 45 sec and extension at 72 °C for 1 min. After completion of 34 cycles, a final extension of 10 min was applied. The PCR products were confirmed by agarose gel electrophoresis using loading star (Dyne Bio) and a 100-bp DNA ladder (Dyne Bio). α-Syn F primer: 5′-TGT AGG CTC CAA AAC CAA GG-3′; R primer: 5′-GCT CCC TCC ACT GTC TTC TG-3′ and hOmi F primer: 5′- GTC GCC GGA TCC ATG CGC TAC ATT-3′R primer: 5′-GAG CTC TCG AGT CAT TCT GTG ACC-3′.

Total RNA was isolated from the mini-pig iliac artery (5mm length) using QIAzol-reagent (QIAGEN, Hilden, Germany), and complementary DNA (cDNA) was synthesized using Quantitect Reverse Transcription (QIAGEN). The cDNA was amplified using AccuPower PCR Premix (Bioneer, Daejeon, Korea). The following primer sequences were used: RAGE, forward 5′-AAG CTT GGA AGG TCC TGA CT-3′ and reverse 5′-ACT TGG TCT CCT TTC CGT TC-3′; HMGB1, forward 5′-CCA TTG GTG ATG TTG CAA AG-3′ and reverse 5′-AGC CTT GAC GAC TCC CTT TT-3′; TNF-α, forward 5′-CCA CCA ACG TTT TCC TCA CT-3′ and reverse 5′-CCA AAA TAG ACC TGC CCA GA-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-GTC GGT TGT GGA TCT GAC CT-3′ and reverse 5′-AGC TTG ACG AAG TGG TCG TT-3′. Relative mRNA levels were determined by comparison to GAPDH mRNA. All PCR products were separated by electrophoresis on 1.5% agarose gels. Size was compared to a 100-bp DNA ladder (DYNE Bio, Seongnam, Korea), and PCR products were visualized using Loading STAR (DYNE Bio).