Total RNA was extracted from the leaves of PA1/PA1 and pa1/pa1 plants at the seedling stage using the TIANGEN kit with DNase I. The RNA was reverse transcribed using PrimeScript RT reagent kit with gDNA Eraser (Takara). Reverse transcription qPCR analysis was conducted with the StepOnePlus System (Applied Biosystems) using TB Green Premix Ex Taq GC (Takara). The reaction procedure was 95 °C for 30 s, 95 °C for 5 s and 60 °C for 30 s for 40 cycles. Actin was used as the internal control gene. All analyses were conducted with four biological replicates. The relative gene expression levels were calculated using the 2 − ΔCt method, and a t-test was performed to compare the results of PA1/PA1 and pa1/pa1 plants.
Tb green premix ex taq gc
Manufactured by Takara Bio
Sourced in Japan
TB Green Premix Ex Taq GC is a qPCR reagent that utilizes TB Green dye for the detection and quantification of DNA sequences. It includes all necessary components for real-time PCR amplification and detection, including a hot-start DNA polymerase, dNTPs, and buffer.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Agencourt dnadvance, by Beckman Coulter (1 mentions)
Superscript 3 first strand kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Dnase treatment, by Promega (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Isogen, by Nippon Gene (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Cfx96 trademark real time pcr detection system, by Bio-Rad (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sa β gal staining kit, by Beyotime (1 mentions)
8 protocols using tb green premix ex taq gc
1
Quantitative RT-PCR Analysis of PA1 Gene Expression
2
AAV Genome Quantification in Mouse Brain
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Transduction efficiency was estimated based on previous reports [80 (link),81 (link)]. After the sleep phenotyping, the brain hemisphere except for the olfactory bulb and cerebellum was collected from the AAV-administrated mouse. Brain DNA was purified using an Agencourt DNAdvance (Beckman Coulter, USA). The copy numbers of both the AAV vector genomes and mouse genomic DNA were quantified with a standard curve generated from known amounts of DNA. Vector genomes per cell were calculated by dividing the copy number of AAV vector genomes by diploid copies of the Tbp gene in the sample. The copy number of the AAV vector genomes and the Tbp gene were determined with WPRE-binding primers (5′-CTGTTGGGCACTGACAATTC-3′, 5′-GAAGGGACGTAGCAGAAGGA-3′) and Tbp-binding primers (5′-CCCCCTCTGCACTGAAATCA-3′; 5′-GTAGCAGCACAGAGCAAGCAA-3′) [70 (link)], respectively. The qPCR protocol was 60 s at 95 °C for preheating (initial denaturation) and 45 cycles from 10 s at 95 °C to 30 s at 60 °C using a TB Green Premix Ex Taq GC (Takara Bio, Japan).
3
qRT-PCR Analysis of miR-1, Bax, and Bcl-2
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To determine miR-1 expression levels, total miRNA was extracted from cells using the miRcute miRNA extraction and isolation kit. Reverse transcription was performed using an miRNA first-strand cDNA synthesis kit (cervical-loop method). To determine Bax and Bcl-2 mRNA levels, total RNA extracted from the cardiomyocytes was reverse transcribed into cDNA. Quantitative real-time PCR was performed using TB Green Premix Ex Taq GC (TaKaRa, Dalian, China). Samples were analyzed in triplicate and GAPDH (or U6) was used as the internal reference gene. The products were predenatured at 95°C for 30 s, denatured at 95°C for 5 s, annealed at 60°C for 30 s, and amplified for 36 cycles. After the reaction, Ct values were obtained determined using the 7500 software program. Data were exported for statistical analysis, and the 2−△△Ct method was used for calculations. Primers that were used are shown in Table 1 .
4
cDNA Synthesis and qRT-PCR Analysis
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cDNA was synthesized using the SuperScript III First Strand kit (Invitrogen) with random hexamer primer, according to the manufacturer’s instructions. qRT-PCR was carried out using TB Green® Premix Ex Taq GC (TaKaRa). Gene expression data were normalized to sigA. Relative gene expression was calculated using the 2−ΔΔCt method. The primers used for the qRT-PCR were described in Table S2.
5
Quantitative Analysis of Inflammation and Oxidative Stress Markers
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Total RNA was extracted using TRIzol® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA quality and quantity were measured using a NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized from the total RNA using the Prime Script RT Reagent Kit (RR036A; Takara Biotechnology Co., LTD., Shiga, Japan). Primers for tumor necrosis factor (TNF) receptor-associated factor 6 (Traf6), myeloid differentiation factor88 (MyD88), TNF, interleukin (IL)1B, Keap1, Nfe2l2/Nrf2, heme oxygenase (Hmox/HO)-1, NAD(P)H quinone oxidoreductase 1 (NQO1), Sqstm1/P62, and β-actin were designed using the Get Prime software (https://gecftools.epfl.ch/getprime ). Real-time Quantitative PCR (RT-qPCR) was conducted using TB Green™ Premix Ex Taq™ GC (RR820A; Takara, Shiga, Japan) on a Light Cycler® 96 System (Roche Life Science, Penzberg, Germany). Relative quantification of gene expression was performed based on the comparative CT (2–ΔΔCt) method. The mRNA expression levels of target genes were normalized to that of the β-actin internal standard. Data were presented as fold changes over the controls, which were shown as 1. The sequences of primers used for RT-qPCR are shown in Table S1 .
6
Quantitative PCR Expression Analysis
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Total RNA was isolated with Isogen (#315-02504, Nippon Gene) and RNeasy Mini Kit (#74104, Qiagen). After DNAse treatment (#M6101, Promega), cDNA was prepared using an iScript cDNA Synthesis Kit (#1708890, BIO-RAD) according to the protocol of the manufacturer. qPCR was performed using TB Green Premix Ex Taq GC (#RR071A, TaKaRa) in a 7500 Real Time PCR System (Applied Biosystems). The following qPCR conditions were employed: one cycle for the initial denaturation stage at 95°C for 30 s, 70 cycles for the PCR stage with denaturation at 95°C for 10 s, and annealing at 60°C for 34 s; the melt curve stage was set according to the instructions for Takara TB Green for the 7500 Real-Time PCR System. The cell counts of the operation samples used in RNA extraction and qPCR are displayed in Supplementary Table S1 Supplementary Table S2
7
Quantitative Analysis of Odontoblast Markers
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Total DPSC RNAs were isolated and extracted by RNAiso plus (TaKaRa, Japan) according to the manufacture’s instruction. Then, the mRNA was reversely transcribed into cDNA by Primescript™ RT master mix (TaKaRa, RR036A, Japan). Real-time PCR was performed with TB Green® Premix Ex Taq™ GC (TaKaRa, RR071A, Japan) and detected by CFX96 Trademark Real-time PCR detection system (Bio-rad, USA). Expression levels of DSPP, DMP1, and β-catenin were examined. All the RT-PCR data are presented as mean ± s.d. for triplicate samples from a representative experiment (n = 3). The primers used in real-time PCR were listed in the supplementary Table 1 .
8
Senescent Cell Evaluation in NPCs
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The senescent cells in NPCs subjected to siRNA and rapamycin were evaluated with SA-β-gal Staining Kit (Beyotime) as suggested in the instruction. The SA-β-gal positive cells were stained into blue and counted in a double-blind manner. In each sample, 10 different non-overlapping elds under 20-fold magni cation were randomly selected, and the SA-β-gal positive ratio was calculated.
RNA extraction and quantitative real-time PCR Total RNA was extracted from NPCs isolated from neonatal, young and middle-aged mice using TRIzol reagent (Invitrogen), and was reverse-transcribed into cDNA utilizing the PrimeScriptTM RT reagent kit (Takara, RR047A, Japan) following the instruction. Gene expression levels of Atg3, Atg5, Atg7 and Beclin1 were quanti ed using the TB Green® Premix Ex Taq™ GC (Takara, RR071A, Japan) on the StepOne Plus Real-Time PCR System (Applied Biosystems Inc., Foster City, CA). In a 20 µl reaction mixture, the amount of cDNA template added was 100ng, and the nal concentration of primer was 0.2μM. The mRNA levels were quantitatively analyzed by 2 -∆∆Ct method and normalized with GAPDH. Sequences of primers utilized in the assays were listed in Table 1.
RNA extraction and quantitative real-time PCR Total RNA was extracted from NPCs isolated from neonatal, young and middle-aged mice using TRIzol reagent (Invitrogen), and was reverse-transcribed into cDNA utilizing the PrimeScriptTM RT reagent kit (Takara, RR047A, Japan) following the instruction. Gene expression levels of Atg3, Atg5, Atg7 and Beclin1 were quanti ed using the TB Green® Premix Ex Taq™ GC (Takara, RR071A, Japan) on the StepOne Plus Real-Time PCR System (Applied Biosystems Inc., Foster City, CA). In a 20 µl reaction mixture, the amount of cDNA template added was 100ng, and the nal concentration of primer was 0.2μM. The mRNA levels were quantitatively analyzed by 2 -∆∆Ct method and normalized with GAPDH. Sequences of primers utilized in the assays were listed in Table 1.
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