The total RNA was extracted from treated and cultured PCa cells. Then, cDNA was synthesized from 500 ng RNA of each sample, 2 µL PrimeScript RT Master Mix (Takara Bio, Otsu, Japan), and RNase-free dH2O (Takara Bio, Otsu, Japan), which was added to a final volume of 10 µL. The total volume (10 µL) of each PCR reaction mixture contained 2.5 µL SYBR Green Supermix (Takara Bio, Otsu, Japan), 1 µL RNase-free dH2O, 1 µL cDNA, and 10 µM of each of forward and reverse primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The sequences of primers are summarized in Table 1 .
Rnase free dh2o
Manufactured by Takara Bio
Sourced in Japan, China
RNase Free dH2O is a high-quality deionized and filtered water designed for use in RNA-based applications. It is processed to remove RNase enzymes, which can degrade RNA molecules. This product is suitable for use in RNA extraction, reverse transcription, and other sensitive RNA-based procedures.
Automatically generated - may contain errors
Lab products found in correlation
Rox reference dye, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rox reference dye 2 50, by Takara Bio (1 mentions)
Dnase 1, by Nippon Gene (1 mentions)
Isogen 2, by Nippon Gene (1 mentions)
Sybr green supermix, by Takara Bio (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Oligo dt primer, by Takara Bio (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Random 6 mers, by Takara Bio (1 mentions)
Primescript rt enzyme mix, by Takara Bio (1 mentions)
Trizol total rna isolation reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green 1, by Takara Bio (1 mentions)
α mem medium, by Thermo Fisher Scientific (1 mentions)
Primescripttm rt reagent kit with gdna eraser perfect real time, by Takara Bio (1 mentions)
Tb green premix ex taqtm 2 tli rnaseh plus, by Takara Bio (1 mentions)
Hoechst 33258 staining kit, by Beyotime (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
7 protocols using rnase free dh2o
1
Quantitative RT-PCR for Gene Expression
2
Quantification of Gene Expression in Chick Palate and Tongue
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The palate and tongue tip were collected from 3-day-old chicks (n = 3). Total RNA was isolated from these tissues with the use of the reagent ISOGEN II (Nippon Gene). Genomic DNA was removed with the use of DNase I (Nippon Gene). The PCR mixture had a total volume of 10 μL and consisted of 5.0 μL 2 × OneStep SYBR reverse transcription PCR (RT-PCR ) Buffer 4 (Takara Bio), 0.4 μL PrimeScript 1 step Enzyme Mix 2 (Takara Bio), 0.4 µL forward primer (10 µM), 0.4 μL reverse primer (10 µM), 0.2 μL ROX Reference Dye II (50 ×) (Takara Bio), 1.0 μL total RNA (100 ng/μL), and 2.6 µL RNase Free dH2O (Takara Bio). The PCR reactions were conducted under the following conditions: 42°C for 5 min, 95°C for 10 s, 40 cycles of (95°C for 5 s and 60°C for 34 s), followed by a melting curve analysis from 60°C to 95°C. The primers used are listed in Table 1 . GAPDH was used as an internal control gene.
3
Total RNA Extraction and cDNA Synthesis
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Total RNA from HEK-293 or SK-N-SH+pEGFP-N1-Basic and HEK-293 or SK-N-SH+pEGFP-N1-CEBPB cells was extracted according to the following procedures. 800 μL Trizol was added to each 6-well plates, and the cell lysate was successively treated with chloroform, isopropanol and 75% ethanol. Total RNA was then dissolved in 30 μL diethyl pyrocarbonate (DEPC)water for several hours and quantified using a UV spectrophotometer. The complementary DNA (cDNA) reaction system included 5 × PrimeScript Buffer 4 μL, PrimeScript RT Enzyme Mix I 1 μL or PrimeScript RT Enzyme Mix I-free (negative control), Oligo dT Primer 1 μL (50 μmol/L), Random 6 mers 1 μL (100 μmol/L), Total RNA (1000 ng) and RNase free dH2O (Takara, Dalian, China). The reaction conditions were 37 °C for 15 min, 85 °C for 5 s, and 4 °C. The real time PCR reaction system included SYBR Green Premix Ex Taq II 10 μL, forward primer and reverse primer 1.6 μL (5 pmol/ μL), ddH20 4.8 μL and cDNA 2 μL. Reaction conditions: pre-denaturation: 95 °C 30 s; denaturation: 95 °C 5 s, 60 °C 30 s, 40 cycles; dissolution curve: 95 °C 15 s, 60 °C 30 s, 95 °C, 15 s.
4
Quantitative RT-PCR Analysis of DMSCs
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Total RNA from DMSCs was extracted using Trizol (Invitrogen, USA), RNA purity was determined (Biodrop, CO, UK), and cDNA was synthesized (Takara, Shiga, Japan). cDNA was synthesized using 5X PrimeScript RT Master Mix (2 μl) and total RNA and RNase Free dH2O in a total volume of 10 μl (TaKaRa, Shiga, Japan). PCR amplification was performed in a 20 μl reaction system containing 2 μl template, 10 μl TB Green Premix Ex Taq II (TaKaRa), 0.4 μl Rox Reference Dye, 6 μl sterile purified water, and 0.8 μl of PCR primers. The primer sequences are shown in Table 1 . Initial denaturation was performed at 95℃ for 30 s and was followed by 40 cycles of denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, dissociation at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. PCR products were identified using 2% agarose gel electrophoresis.
5
Total RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol total RNA isolation reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Specific primers from Invitrogen; Thermo Fisher Scientific, Inc., (Shanghai, China) were used for transcript detection: Actin forward, 5′-CTCCATCCTGGCCTCGCTTGT-3′ and reverse, 5′-GCTGTCACCTTCACCGTTCC-3′; SPARC forward, 5′-GGAAGAAACTGTGGCAGAGG-3′ and reverse, 5′-ATTGCTGCACACCTTCTCAA-3′. qPCR (5X PrimeScript buffer 2 µl, PrimeScript RT Enzyme Mix 1 0.5 µl, Oligo Dt Prime 0.5 µl, Random 6 mers 0.5 µl, total RNA and RNase Free dH2O up to 10 µl; Takara Bio, Inc., Otsu, Japan) was performed with SYBR Green I (Takara Bio, Inc.). The progression consisted of 40 cycles (95°C for 15 sec and 60°C for 1 min) subsequent to an initial denaturation step (95°C for 10 min). The mean number of three independent analyses for each gene and sample was calculated and normalized to the endogenous GAPDH (12 (link)) reference control gene actin using the 2−ΔΔCq method (13 (link)).
6
Frondoside A Inhibits Bladder Cancer
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Triterpene glycoside Frondoside A (extracted from Cucumaria frondosa, purity > 98% determined by HPLC) was purchased from Kerafast (Boston, MA, USA). Cell Counting kit-8 (CCK-8) was purchased from US EVERBRIGHT® Inc. (Suzhou, Jiangsu, China). Hoechst-33258 Staining Kit, Annexin-V-FITC Apoptosis Detection Kit, and Cell Cycle and Apoptosis Analysis Kit were obtained from Beyotime (Zhenjiang, Jiangsu, China). α-MEM medium, fetal bovine serum (FBS), PBS, penicillin (100 Units/mL), streptomycin (100 μg/mL), 0.25% Trypsin (1X), and Nonessential Amino Acid Solution (NAAS, 100X) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time), TB Green® Premix Ex TaqTM II (Tli RNaseH Plus), and RNase Free dH2O were obtained from TaKaRa Bio (TaKaRa, Dalian, China). Chemotherapetic agent Epirubicin hydrochloride (EPI), a commonly used chemotherapy drugs to treat bladder cancer in the clinic, was purchased from Aladdin (Shanghai, China).
7
Quantitative Gene Expression Analysis by RT-qPCR
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RNA from all 26 samples was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using 4 μL total RNA, 4 μL 5X PrimeScript buffer, and 12 μL RNasefree dH 2 O in a total volume of 20 μL (TaKaRa, Shiga, Japan). PCR amplification was performed in a 20-μL system containing 2 μL cDNA, 10 μL 2X SYBR Premix EX Taq II buffer (TaKaRa), 0.4 μL Rox Reference Dye, 6 μL RNase-free dH 2 O, and 0.8 μL of each 10 μM primer. The thermal cycling conditions included pre-denaturation at 95°C for 30 s, 40 cycles of denaturation at 95°C for 10 s, and annealing at 60°C for 30 s. β-actin was used as an internal control as previously reported. PCR products were identified using 2% agarose gel electrophoresis. Primer sequences and amplification product sizes are shown in Table 2.
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