Total RNAs were extracted from the whole ipsilateral cortex of sham-operated (control) or photothrombosis (PT) mice at day 1, 3, 7, 14, 28 post-stroke using RNAIso-PLUS (Takara Bio, Japan). RNA (0.5 μg) was reverse transcribed to produce cDNA using ReverTra Ace® qPCR RT Kit with gDNA remover (TOYOBO, Japan). Real-time PCR was performed using THUNDERBIRD® SYBR® qPCR Mix (TOYOBO, Japan). All real-time PCR assays were performed in biological triplicate using 7300 Real-Time PCR System (Applied Biosystems). Primers in the study were listed on Supplementary Table S1 . For standardization of relative mRNA expression, 18 s ribosomal primers were used. The results of cycle threshold values (Ct values) were calculated by the ΔΔCt method to obtain the fold differences. Five brains were used for real-time PCR analysis at each time point. Data were expressed as fold changes vs. control (sham-operated mice) ± SEM. The asterisks indicate a statistically significant difference from the control value (*p < 0.05, **p < 0.01 vs. control, Dunnett’s multiple comparison test of Biocunductor in R studio).
Revertra ace qpcr rt kit with gdna remover
Manufactured by Toyobo
Sourced in Japan
The ReverTra Ace qPCR RT Kit (with gDNA remover) is a laboratory equipment product designed for reverse transcription and real-time quantitative PCR (qPCR) analysis. The kit includes a reverse transcriptase enzyme and a genomic DNA (gDNA) removal solution to facilitate the synthesis of cDNA from RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Rnaprep pure plant kit, by Tiangen Biotech (2 mentions)
Lightcycler faststart dna master sybr green 1 kit, by Roche (2 mentions)
Lightcycler 96 real time pcr detection system, by Roche (2 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Thunderbird sybr qpcr mix, by Toyobo (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Uv 2550 spectrophotometer, by Shimadzu (1 mentions)
Lymphocyte separation medium, by Solarbio (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Qiagen (1 mentions)
Thunderbird next sybr qpcr mix, by Toyobo (1 mentions)
Brilliant 3 ultra fast sybr green qpcr master mixes, by Agilent Technologies (1 mentions)
Trisure, by Nippon Genetics (1 mentions)
Plant dna mini kit, by Aidlab (1 mentions)
Rotor gene 6000 machine, by Qiagen (1 mentions)
7 protocols using revertra ace qpcr rt kit with gdna remover
1
Stroke-induced Transcriptomic Changes
2
Peripheral Blood Mononuclear Cell Isolation and RNA Extraction
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Mononuclear cells were isolated from 5 ml of peripheral blood (before and after moving to the plateau, 3,700 m) by using lymphocyte separation medium (Solarbio, Beijing, China), as previously described (Chen et al., 2016 (link)). Total RNA was extracted from 107 mononuclear cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol and then quantified using a UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). cDNA was synthetized from approximately 0.5 µg of total RNA using a ReverTra Ace®qPCR RT kit with gDNA Remover (TOYOBO, Osaka, Japan).
3
Cotton Root RNA Extraction and qPCR Analysis
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The total RNA of cotton roots were extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). The quantity and quality were determined by a NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The cDNA was reverse transcribed with the ReverTra Ace qPCR RT Kit (with gDNA remover) (Toyobo, Osaka, Japan). All the operational procedures followed the manufacturer's protocols. The qPCR analysis was performed using a LightCycler FastStart DNA Master SYBR Green I kit (Roche, Basel, Switzerland) on a LightCycler96 Real-Time PCR detection system (Roche). For internal reference gene, UBQ7 was used, which was stably expressed in cotton plants and not affected by treatments and genotypes. All RT-qPCR analyses were performed with three biological replicates and each analysis was carried out in three technical RT-qPCR replicates. Statistical analysis was conducted with biological replicates with mean values of three technique replicates. 2 -△CT method was used to further study the data from real-time PCR amplification (Livak and Schmittgen 2001) (link). The primers were designed using Primer-BLAST (Ye et al. 2012) (link), listed in Supplementary Table S1.
4
Quantitative Analysis of Gene Expression
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Hypothalami isolated from mice were placed in RNA Later (QIAGEN, USA). Total RNA from the tissues of mice and cultured cells was extracted with TRIsure (NIPPON Genetics, Japan) and subjected to reverse transcription using the ReverTra Ace qPCR RT kit with gDNA remover (TOYOBO, Japan). Relative gene expression was measured by Mx3000/Mx3005P (Agilent Technologies, USA) or CronoSTAR96 (Takara Bio, USA) with Brilliant III Ultra-Fast SYBR Green QPCR Master Mixes (Agilent Technologies) or THUNDERBIRD Next SYBR qPCR Mix (TOYOBO) and 0.2 μM of primers. The sequences of each primer are listed in Additional file 1 : Table S1. Gene expression was normalized with 18S ribosomal RNA (Tanaka et al. 2021 (link)).
5
Quantitative Analysis of Gene Expression
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Total DNA was extracted from plant leaves using the Plant DNA Mini Kit (Aidlab, Beijing, China) according to the manufacturer's instructions. Total RNA was extracted from different tissues using the RNAprep Pure Plant Kit (Tiangen). Total RNA (500 ng) was reverse transcribed using the ReverTra Ace qPCR RT Kit (with gDNA remover) (Toyobo, Osaka, Japan). qPCR analysis was performed using a LightCycler FastStart DNA Master SYBR Green I kit (Roche, Basel, Switzerland) on a LightCycler96 Real‐Time PCR detection system (Roche). Three independent biological replicates were used for each analysis with at least three technical replicates each. The relative expression levels were evaluated using the comparative cycle threshold method according to Livak and Schmittgen (2001 ). GhUBQ7 (Tan et al., 2013 ) was used as the internal control, which was stably expressed in cotton plants and not affected by treatments and genotypes. The gene‐specific primers were designed using Primer Premier 5 software. All primers are listed in Table S6 .
6
Osteogenic and Macrophage Gene Expression in Transplanted GAMs
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At 1- and 2-weeks post-transplantation, the transplanted GAMs were harvested and pulverized using a homogenizer (MP-Biomedicals, Tokyo, Japan). Total RNAs were extracted using TRI Reagent. Next, qPCR was employed to detect the mRNA expressions of the osteogenic (bmp4 and osteocalcin) and macrophage (f4/80 and cd206) genes in the specimens. The ReverTra Ace® qPCR RT Kit with gDNA Remover (Toyobo) was used for cDNA synthesis. Then, qPCR was performed using SYBR green and gene-specific primers on an Mx3000p real-time PCR system. Table 1 shows the rat-specific primer sets; glyceraldehyde-3-phosphate dehydrogenase (gapdh) was employed as the internal standard.
7
Quantitative Gene Expression Analysis
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Total RNA was extracted from the pituitary using the Genezol™ reagent following the product instruction. The RNA concentration and purity were measured using a spectrophotometer at 260 and 280 nm. The RNA purification and first-strand cDNA synthesis were conducted using the ReverTraAce®qPCR RT kit with gDNA Remover (Toyobo, Japan) from the 20 ngµL -1 of total RNA. The quantitative real-time PCR (qPCR) method was used to compare the gene expression level of FSH- and LH- between treatments. The qPCR reaction was conducted in Rotor-Gene 6000 machine (Corbett, USA) with the -actingene as the calibrator. The amplification was performed using a sensifast SYBR® NO-ROX kit (Bioline, UK) with a total of 20 µL. The qPCR program was optimized in the preliminary study, i.e., pre-denaturation at 95°C for two minutes, 40 cycles of amplification for denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 10 seconds. The qPCR primers were designed based on the FSH-, LH-, -actin mRNA sequence of the Pristolepis fasciata, since the sequence data of P. grootii is not available yet in the Genbank. The primers sequence is presented in Table1.
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