Total RNA was extracted with an AccuPrep® Universal RNA Extraction Kit (Bioneer Corp., Daejeon, Republic of Korea), and complementary DNA (cDNA) was synthesized with PrimeScript RT Master Mix (Takara Bio, Shiga, Japan), following the manufacturer’s protocol. Then, qRT-PCR was carried out using TB Green® and QuantStudio 3 (Thermo Fisher Scientific Inc., Waltham, MA, USA). The primers were purchased from Bioneer Corp.
Tb green
Manufactured by Thermo Fisher Scientific
Sourced in United States
TB Green is a fluorescent dye used in real-time PCR (polymerase chain reaction) assays for the detection and quantification of DNA. It binds to double-stranded DNA and emits a fluorescent signal, which can be measured to determine the amount of target DNA present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt master mix, by Takara Bio (3 mentions)
Quantstudio 3, by Thermo Fisher Scientific (2 mentions)
Accuprep universal rna extraction kit, by Bioneer (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Accutarget qpcr screening kit, by Bioneer (1 mentions)
Steponeplus pcr system, by Thermo Fisher Scientific (1 mentions)
Arevertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
5 protocols using tb green
1
Total RNA Extraction and qRT-PCR Analysis
2
Comprehensive Gene Expression Analysis
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Total RNA was extracted with AccuPrep Universal RNA Extraction kit (Bioneer, Daejeon, Korea), and cDNA was synthesized with PrimeScript RT Master mix. Then, quantitative real-time PCR was carried out using TB Green and QuantStudio 3 (Thermo fisher scientific, Massachusetts, USA). The primers were purchased from Bioneer (Accutarget qPCR Screening kit, Bioneer, Daejeon, Korea) (Supplementary Table 2 ).
3
Quantitative RT-PCR Analysis of Gene Expression
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Cells were lysed, and total RNA was extracted using Trizol reagent. cDNA was made from total RNA using oligo (dT) primers with a a RevertAid First Strand cDNA Synthesis Kit (Cat# K1621; Fermentas, Waltham, MA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the TB Green and StepOnePlus PCR system (Applied Bio-systems). The following conditions were used for amplification of fragments: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Relative quantification was expressed as 2−Ct, where –Ct is the difference between the main Ct value of triplicates of the sample and that of an endogenous GAPDH mRNA control. The Gene-specific primer sequences used are listed in the Supplemental Material (Additional file 1 : Table S1).
4
Quantifying Glucose Metabolism and mTOR Signaling in T Cell Subsets
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Total RNA was extracted from CD4+ Tregs and Teffs using the RNeasy Micro kit (Qiagen) and then reverse transcribed into complementary DNA with Prime Script RT Master Mix (Takara) following the manufacturer's instructions. Genes related to glucose metabolism and mTOR signal mRNA levels were quantified using TB Green and a 7500 Real‐Time PCR system (Applied Biosystems, Life Technologies). The primer sequences are shown in in Table 2 . The relative expression of these target genes normalized by β‐actin was calculated as 2−ΔCt.
5
Evaluating Glucose Metabolism Gene Expression
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Total RNA was isolated from CD4+ Treg and Teff cells using RNeasy Micro kit (Qiagen, Dusseldorf, Germany), and then reverse transcribed to cDNA with Prime Script RT Master Mix (Takara, Otsu, Japan) according to the manufacturer’s instructions. Genes related to glucose metabolism mRNA levels were quantified with TB Green and a 7500 Real-Time PCR system (Applied Biosystems; Life Technologies, Grand Island, NY, USA). The primer sequences were in Table 1 . The relative expression of these target genes normalized by β-actin was calculated as 2−ΔCt.
List of PCR primer sequences.
| Gene name | Upstream primers (5′→3′) | Downstream primers (5′→3′) |
|---|---|---|
| Glut1 | TTGGCTCCGGTATCGTCAAC | GCCAGGACCCACTTCAAAGA |
| Glut3 | GCTCTCTGGGATCAATGCTGTGT | CTTCCTGCCCTTTCCACCAGA |
| HIF-1α | CCATTAGAAAGCAGTTCCGC | TGGGTAGGAGATGGAGATGC |
| TPI | AGGCATGTCTTTGGGGAGTC | AGTCCTTCACGTTATCTGCGA |
| GPI | AGGCTGCTGCCACATAAGGT | AGCGTCGTGAGAGGTCACTTG |
| LDHα | CCAGCGTAACGTGAACATCTT | CCCATTAGGTAACGGAATCG |
| ENO1 | TCATCAATGGCGGTTCTCA | TTCCCAATAGCAGTCTTCAGC |
| PKM2 | GCCGCCTGGACATTGACTC | CCATGAGAGAAATTCAGCCGAG |
| mTOR | ATGCTTGGAACCGGACCTG | TCTTGACTCATCTCTCGGAGTT |
| p70S6K | AGCCAAAGATCACATAGTGGT | GGCAGATATTCAACTTTGTCCA |
| AKT | CTGAGATTGTGTCAGCCCTGGA | CACAGCCCGAAGTCTGTGATCTTA |
| 4E-BP1 | TCACACTCAGACTCCGAGA | GAATGCCCTTCCTTAGCAA |
| RHEB | TTGTGGACTCCTACGATCCAA | GGCTGTGTCTACAAGTTGAAGAT |
| TSC1 | ATAGCTGTTACCTCGACGAGT | TGCAGGGAGACCTCTATGTCC |
| β-actin | GAGCTACGAGCTGCCTGACG | GTAGTTTCGTGGATGCCACAG |
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