The procedure used in this study has been published previously (27 (link)–29 (link)); gene expression levels of Ly6G+CD244+ and Ly6G+CD244− cells were assessed by quantitative real-time (q)PCR with the 2ΔCT method for relative quantitation of target genes to an internal control. Total RNA was extracted from the cells with TRIzol reagent (Invitrogen) and reverse transcribed into complementary DNA (cDNA) using a High-Capacity cDNA Reverse Transcription kit (Applied Biosciences). qPCR was conducted on a Takara Thermal Cycler Dice Real-time System (Takara Bio Inc.) with specific primers (Table I ) and KOD SYBR qPCR Mix (Toyobo) according to the manufacturer's instructions. The thermal cycling conditions consisted of 95°C for 30 sec, 40 cycles of 95°C for 5 sec, and 60°C for 30 sec. Amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control.
Takara thermal cycler dice real time system
Manufactured by Takara Bio
The Takara Thermal Cycler Dice Real-time System is a laboratory instrument designed for real-time PCR analysis. It provides precise temperature control and real-time detection capabilities for DNA amplification and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Goscript reverse transcription system kit, by Promega (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Kod sybr qpcr mix, by Toyobo (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Evoscript reverse transcriptase, by Roche (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (1 mentions)
Taqman fast cells to ct kit, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Ribospin 2 kit, by GeneAll (1 mentions)
Topreal qpcr 2 premix kit, by Enzynomics (1 mentions)
Topreal qpcr 2x premix kit, by Enzynomics (1 mentions)
4 protocols using takara thermal cycler dice real time system
1
Quantitative Real-Time PCR Analysis of Immune Cell Subsets
2
Quantitative Real-Time PCR Protocol for Gene Expression Analysis
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Total RNA was extracted and reverse transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher, 4399003). Real-time quantitative RT-PCR was performed using the Taqman PreAmp Master Mix (Thermo Fisher, 4391128), Taqman Fast Universal PCR Master Mix (Thermo Fisher, 4366072), and a QuantStudio 3 Real-Time PCR System (Thermo Fisher). In some experiments, total RNA was extracted using the RNeasy Micro Kit (Qiagen, 74004) and reverse transcribed using the EvoScript Reverse Transcriptase (Roche, 07912315001). Real-time quantitative RT-PCR was performed using the TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara, RR820A) and a Takara Thermal Cycler Dice Realtime System (Takara). Relative expression was calculated for each gene by the comparative CT method and with Actb for normalization. Probes were shown in Supplementary Fig. 2 .
3
Quantitative RT-PCR Analysis of RNA Extraction
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Total RNA was extracted from cells that were pretreated with SA (100, 200, and 400 μM) followed by treatment with 50 μM of 6-OHDA using the Ribospin™ II kit (GeneAll Biotechnology Co., LTD, Seoul, Korea). After measuring the total RNA concentration using a UV/Vis Nano Spectrophotometer (MicroDigital Co., Ltd., Sungnam, Korea), 1 μg of total RNA from each group was reverse transcribed to synthesize cDNA using the GoScript Reverse Transcription System Kit (Promega Co., Madison, WI, USA). qRT-PCR was performed using a TaKara Thermal Cycler Dice Real-Time System (Takara Bio. Inc., Shiga, Japan) using the TOPreal™ qPCR 2× PreMIX Kit (Enzynomics Co., Daejeon, Korea). The primer sequences are as shown in Table 1 .
4
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated by commercially available kit (New England Biolabs Inc., Ipswich, MA, USA). The reverse transcription with 1 μg of total RNA was performed by the GoScript Reverse Transcription System Kit (Promega Co., Madison, WI, USA). qRT-PCR was performed using the TaKara Thermal Cycler Dice Real Time System (Takara Bio. Inc., Shiga, Japan) with the TOPreal™ qPCR 2X PreMIX Kit (Enzynomics Co., Daejeon, Korea). The primer sequences are shown in Table 1 .
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