Thermal cycler dice real time system 2

NIH3T3 cells were seeded into the wells of 6-well plates (at a density of 2 × 10⁵ cells/well). After culturing for 24 h, the cells were treated for 3 h or 24 h with betanin or quercetin at different concentrations. After the medium was removed, total RNA was isolated with the Isogen Ⅱ (Nippon Genetics Co. Ltd., Tokyo, Japan) reagent. Complementary DNA (cDNA) templates were generated using the PrimeScript^TM RT reagent kit with gDNA Eraser (Takara Bio Inc., Kusatsu, Japan). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using TB Green Premix Ex Taq (TliRNaseH Plus, Takara Bio Inc., Kusatsu, Japan) in a Thermal Cycler Dice Real Time System Ⅱ (Takara Bio Inc., Kusatsu, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as an endogenous control for RT-qPCR. The fold change of the mRNA expression was calculated using the standard curve method. The primer sequences used are listed in Table S1.

The cells were lysed using ISOGEN (NIPPON GENE, Tokyo, Japan), and mRNA was isolated using chloroform and isopropyl alcohol. The mRNA was treated with a PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan) to synthesize the cDNAs. The mRNA levels were quantified using quantitative PCR (qPCR) of cDNA with SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara) and the Thermal Cycler Dice Real Time System II (Takara) with the ΔΔCt method. For qPCR, primer sequences for GAPDH and fibronectin were taken from previously published sequences, and the primers were purchased from Hokkaido System Science (Hokkaido, Japan).
The sequences of the PCR primers were GAPDH: forward, 5′-GAGTCAACGGATTTGGTCGT-3′ and reverse, 5′-TTGATTTTGGAGGGATCTCG-3′; fibronectin: forward, 5′-AAACCAATTCTTGGAGCAGG-3′ and reverse, 5′-CCATAAAGGGCAACCAAGAG-3′; and collagen type I, alpha 1 chain (COL1A1): forward, 5′-CAGCCGCTTCACCTACAGC-3′ and reverse, 5′-TTTTGTATTCAATCACTGTCTTGCC-3′. The data were normalized relative to GAPDH.

For disruptants tua1-A, tua2-A, tub1-B, and tub2-A, semi-quantitative real-time PCR was performed by using TB Green Premix Ex Taq GC (Perfect Real Time) and a Thermal Cycler Dice Real-Time System II (Takara, Japan) in accordance with the manufacturer’s instructions.

Reverse-transcription and real-time PCR were performed (n = 10) as described previously (Hatasa et al., 2021 (link)). Briefly, 500 ng of total RNA was reverse-transcribed to cDNA using the PrimeScriptTM RT Master Mix (TaKaRa Bio). Real-time PCR was performed using the Thermal Cycler Dice^® Real Time System II (TaKaRa Bio). PCR mixtures were prepared using TB Green Premix Ex TaqTM II (TaKaRa Bio). All procedures were performed following the manufacturer’s instructions. Rn18s was used as the reference gene for normalization. The PCR primers used in this study are listed in Supplementary Table S1.

Total RNA was extracted from cells grown in MA5 minimal medium to an OD₇₅₀ of 2.0 using a TRIzol® plus RNA purification kit (Ambion), and remaining DNA was digested using a TURBO DNA-free kit (Ambion) according to the manufacturer’s instructions. First-strand cDNA was synthesized using a PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time, TaKaRa) and an RT primer mix containing oligo (dT)₁₈ and random hexamers. Real-time PCR (95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s) to quantify PeUMPS cDNA was performed using the primers Umps_QRT_F (5′-TCTTCGACAAGTGGGATGATGG-3′; position 908–930 of the 1597-bp UMPS cDNA) and Umps_QRT_R (5′-TGTTACCGATGTCCGCAAAC-3′; position 993–1012 of the 1597-bp UMPS cDNA) on a TaKaRa Thermal Cycler Dice® real-time system II using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus, TaKaRa) according to the manufacturer’s instructions. The levels of 18S rRNA were also determined as an internal control using the primers 18SrRNA_QRT_F (5′-GGATCAATTGGAGGGCAAGT-3′; position 627–646 of the 1899-bp 18S rRNA) and 18SrRNA_QRT_R (5′-GCCCGAAATCCAACTACGAG-3′; position 713–732 of the 1899-bp 18S rRNA). The melting curves for each PCR product were determined by measuring the decrease in fluorescence with increasing temperature from 60 to 95 °C. Real-time PCR without a template was also performed in each experiment as a negative control.