Total RNA was isolated using the NucleoSpin RNA II extraction kit (Macherey-Nagel GmbH & Co. KG, Dueren, Germany) and used for cDNA synthesis with a ReverTra Ace-α- reverse transcriptase kit (Toyobo Co., Ltd., Osaka, Japan); both kits were used according to the manufacturers' instructions. The amount of standard cDNA was determined photometrically. The cDNA was used for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using SsoAdvanced SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). PCR was performed using the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) with a C1000 Thermal Cycler (Bio-Rad Laboratories, Inc.). PCR reactions for HIF-1α, eNOS, and GAPDH were initiated with a denaturing step at 95°C for 3 min, followed by 40 cycles at 95°C for 10 sec, 58°C for 10 sec, and 72°C for 20 sec. A melting curve (ramping from 65°C to 95°C) was performed following RT-PCR to test for the presence of primer dimers. When primer dimer formation was detected, the PCR was repeated using a separate cDNA aliquot. Each measurement was repeated three times, and the values were used to calculate the ratio of HIF-1α to GAPDH, with the control set at a value of 1.0 to serve as a standard.
Revertra ace α reverse transcriptase kit
Manufactured by Toyobo
Sourced in Japan
The ReverTra Ace-α- reverse transcriptase kit is a lab equipment product manufactured by Toyobo. It is a reverse transcriptase enzyme used in the process of converting RNA into complementary DNA (cDNA).
Automatically generated - may contain errors
Lab products found in correlation
Sybr green mix kit, by Toyobo (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Ssoadvanced sybr green supermix, by Bio-Rad (1 mentions)
Nucleospin rna 2 extraction kit, by Macherey-Nagel (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Iq5 real time pcr system, by Bio-Rad (1 mentions)
Sybr green pcr master mix, by Toyobo (1 mentions)
Cfx connect real time pcr system, by Bio-Rad (1 mentions)
4 protocols using revertra ace α reverse transcriptase kit
1
Quantitative RT-PCR Analysis of HIF-1α and eNOS
2
Quantification of Gene Expression via RT-PCR
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The cells (2 × 104) were seeded in 24-well plates and treated with various concentrations of MSU crystals (0.1, 0.2, and 0.3 mg/mL) for 24 h. The total RNA was extracted using a TRIzol reagent, and complementary DNA (cDNA) was synthesized using a ReverTra Ace-α-reverse transcriptase kit (Toyobo, Osaka, Japan). The cDNA was then analyzed by real-time RT-PCR (Bio-Rad iQ5 Real-Time PCR System, Bio-Rad, Hercules, CA, USA) using an SYBR Green Mix kit (Toyobo, Osaka, Japan). The PCR amplification consisted of an initial denaturation at 95 °C for 15 min, followed by 40 cycles of 9 °C for 5 s, 55–65 °C for 30 s, and 72 °C for 15 s. The relative expression of each gene was analyzed using the ΔΔCT method.
3
Evaluating CKD-WID Effects on Osteoclastogenesis
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Cells were seeded in 24-well plates at 1 × 104 cells and treated with sRANKL (100 ng/mL) and MSU crystals (0.1 mg/mL) for 4 days. Various concentrations of CKD-WID (0.5, 1.0, and 3.0 µM) were added to the medium, and cells were further cultured for 4 days. Total RNA of cells was extracted with TRIzol Reagent (Gibco BRL, Grand Island, NY, USA). Complementary DNA was synthesized from 1 μg of RNA using a ReverTra Ace-α- reverse transcriptase kit (Toyobo, Osaka, Japan) in a reaction condition of incubation at 37 °C for 15 min, 50 °C for 5 min, and 98 °C for 5 min.
RT-PCR was performed using the Mini Option TM Real-time PCR system (Bio-Rad, Hercules, CA, USA) with SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) according to the manufacturers’ instructions. The PCR amplification reaction (total volume of 20 µL) contained 2 µL of cDNA, 10 µL of SYBR® Green Realtime PCR Master Mix, 10 pmol/L of each primer, and 6.4 µL of distilled water. The reactions were carried out with an initial denaturation at 95 °C for 15 min, followed by 40 cycles of 95 °C for 5 s, 55–62 °C for 30 s, and 72 °C for 15 s. All reactions were run in triplicate.
RT-PCR was performed using the Mini Option TM Real-time PCR system (Bio-Rad, Hercules, CA, USA) with SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) according to the manufacturers’ instructions. The PCR amplification reaction (total volume of 20 µL) contained 2 µL of cDNA, 10 µL of SYBR® Green Realtime PCR Master Mix, 10 pmol/L of each primer, and 6.4 µL of distilled water. The reactions were carried out with an initial denaturation at 95 °C for 15 min, followed by 40 cycles of 95 °C for 5 s, 55–62 °C for 30 s, and 72 °C for 15 s. All reactions were run in triplicate.
4
Osteoclastogenesis Regulatory Genes Analysis
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Cells (2 × 104/well) were seeded in 24-well plates and pre-incubated with sRANKL (100 ng/ml) for 4 days and then cotreated with CpG-ODN (1 μM) for 2 days. Total RNA was extracted from cells using TRIzol Reagent (Gibco BRL, Grand Island, USA), and complementary DNA (cDNA) was synthesized using a ReverTra Ace-α-reverse transcriptase kit (Toyobo, Osaka, Japan). cDNA was subjected to real time PCR using SYBR Green Mix kit (Toyobo, Osaka, Japan) and the CFX Connect real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturers' instructions.
Primers, synthesized at Bionics Company (Seoul, Korea), were as follows: TRAP, forward 5′- AAG GCG AGA GAT TCT TTC CCT G-3′, reverse 5′-ACT GGG GAC AAT TCA CTA GAG C-3′; cathepsin K, forward 5′- CAG CAG AAC GGA GGC ATT GA-3′, reverse 5′-CCT TTG CCG TGG CGT TAT AC-3′; carbonic anhydrase II, forward 5′- CAT TAC TGT CAG CAG CGA GCA-3′, reverse 5′-GAC GCC AGT TGT CCA CCA TC-3′; NFATc1, forward 5′-CTC GAA AGA CAG CAC TGG AGC AT-3′, reverse 5′-CGG CTG CCT TCC GTC TCA TAG-3′; c-Fos, forward 5′-ACC ATG ATG TTC TCG GGT TTC AA-3′, reverse 5′-GCT GGT GGA GAT GGC TGT CAC-3′; A20, forward 5′-TGCCCAGTCTGTAGTCTTCG-3′, reverse 5′-AGTTGTTCAGCCATGGTCCT-3′; and GAPDH, forward 5′-TGC ACC ACC AAC TGC TTA-3′, reverse 5′- GGA TGC AGG GAT GAT GTT C-3′. The relative expression of each gene was analyzed using the 2–ΔΔCT method.
Primers, synthesized at Bionics Company (Seoul, Korea), were as follows: TRAP, forward 5′- AAG GCG AGA GAT TCT TTC CCT G-3′, reverse 5′-ACT GGG GAC AAT TCA CTA GAG C-3′; cathepsin K, forward 5′- CAG CAG AAC GGA GGC ATT GA-3′, reverse 5′-CCT TTG CCG TGG CGT TAT AC-3′; carbonic anhydrase II, forward 5′- CAT TAC TGT CAG CAG CGA GCA-3′, reverse 5′-GAC GCC AGT TGT CCA CCA TC-3′; NFATc1, forward 5′-CTC GAA AGA CAG CAC TGG AGC AT-3′, reverse 5′-CGG CTG CCT TCC GTC TCA TAG-3′; c-Fos, forward 5′-ACC ATG ATG TTC TCG GGT TTC AA-3′, reverse 5′-GCT GGT GGA GAT GGC TGT CAC-3′; A20, forward 5′-TGCCCAGTCTGTAGTCTTCG-3′, reverse 5′-AGTTGTTCAGCCATGGTCCT-3′; and GAPDH, forward 5′-TGC ACC ACC AAC TGC TTA-3′, reverse 5′- GGA TGC AGG GAT GAT GTT C-3′. The relative expression of each gene was analyzed using the 2–ΔΔCT method.
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