Total RNA was isolated from 100 mg Arabidopsis sp. leaves and roots and from whole seedlings for KO/KD expression studies using Nucleospin Plant RNA kit (Macherey-Nagel, Düren, Germany). First-strand cDNA synthesis of 1 μg of total RNA was carried out in 20 μL with RevertAid M-MuLV Reverse Transcriptase (Applied Biosystems by Thermo Fischer Scientific, Vilnius, Lithuania), using random hexamers. Real-time PCR was carried out with the ABI 7900 Fast Real Time System (Applied Biosystems, Foster City, CA, USA) with the following protocol: 45 cycles at 95 °C for 15 s, followed by 60 °C for 1 min. The normalized relative transcript levels were obtained by the 2−ΔCt method [63 (link)]. At least two biological replicates were performed for each gene tested.
Abi7900 fast real time system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI7900 Fast Real-Time System is a laboratory instrument designed for real-time PCR (Polymerase Chain Reaction) analysis. It provides fast and accurate detection and quantification of target DNA sequences. The system utilizes fluorescence-based detection technology to monitor the amplification of DNA samples in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (14 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (7 mentions)
Sybr pcr master mix reagent kit, by Takara Bio (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Sybr premix ex taq kit, by Takara Bio (4 mentions)
Revertaid m mulv reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Nucleospin rna plant kit, by Macherey-Nagel (2 mentions)
Sybr green qpcr master mixes, by Thermo Fisher Scientific (2 mentions)
Maxima sybr green qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Genejet plant rna purification mini kit, by Thermo Fisher Scientific (1 mentions)
Sybr master mix, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Bullet blender, by Next Advance (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Genejet plant rna purification kit, by Thermo Fisher Scientific (1 mentions)
Trizol protocol, by Thermo Fisher Scientific (1 mentions)
19 protocols using abi7900 fast real time system
1
qRT-PCR Analysis of Arabidopsis Transcripts
2
Quantitative Gene Expression Analysis in Arabidopsis
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Total RNA was isolated from 100 mg Arabidopsis seedling or different plant organs using GeneJET Plant RNA Purification Mini Kit (Thermo Scientific, K0801). 5 of RNA was treated with TURBO DNA‐free™ Kit (Thermo Fisher Scientific) and then 1 µg RNA was used for cDNA Synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real‐time PCR was carried out with the ABI 7900 Fast Real‐Time System (Applied Biosystems) using SYBR Green qPCR Master Mixes (Thermo Scientific) following the manufacturer's instructions. Relative transcript levels were standardized to UBC18 (AT5G42990), ACT2 (AT3G18780) or 18S RNA as reference genes and calculated by the 2−∆Ct or 2−ΔΔCt method (Livak & Schmittgen, 2001 (link)). At least two biological replicates were made.
3
Arabidopsis Transcriptome Analysis by Real-Time PCR
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Total RNA was isolated from 100 mg Arabidopsis seedlings, using a Nucleospin Plant RNA Kit (Macherey-Nagel). Isolated RNA was DNase treated with TURBO DNA-free™ Kit (Invitrogen/Thermo Fisher Scientific), and first-strand cDNA synthesis of 1 µg of total RNA was carried out with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems/Thermo Fisher Scientific), using random hexamers. Real-time PCR was carried out with the ABI 7900 Fast Real Time System (Applied Biosystems) with the following protocol: 40 cycles at 95 °C for 15 s, 60 °C for 60 s, using Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific). GAPDH2 (AT1G13440) was used as reference gene. The normalized relative transcript levels were calculated with the 2−ΔΔCt method (Czechowski et al., 2005 (link)).
4
RNA Extraction and qPCR from Frozen Tissue
5
Quantifying mtDNA-encoded OXPHOS Subunits
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The mRNA levels for mtDNA-encoded OXPHOS subunits, p53 pathway were analyzed using SYBR PCR Master Mix reagent kits (Takara, Tokyo, Japan) according to the manufacturer’s instructions. All oligo nucleotide primers were synthesized by Invitrogen (Shanghai, China). All real-time PCR reactions were carried out on an ABI7900 Fast Real-Time System (Applied Bio systems, Foster City, CA, USA) according to the manufacturer’s instructions. All experiments were repeated at least three times. Primer sequences are shown in Table S3 .
6
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from cells using TRIzol reagent (Invitrogen Life Technologies Co, USA) according to manufacturer instructions. The concentration of total RNA was measured by NanoDrop 2000 (Thermo Fisher Scientific, USA). One microgram of total RNA was reverse transcribed into cDNA using the Prime Script™ RT Reagent Kit with gDNA Eraser (Perfect Real Time, Takara, Japan). The genomic DNA was removed under 42 °C for 2 min and cDNA was synthesized under 37 °C for 15 min and 85 °C for 30 s. The qRT-PCR was performed on an ABI7900 Fast Real-Time System (Applied Bio systems, USA) using a SYBR Premix Ex Taq™ Kit (Takara, Japan). Data analysis was done by using the comparative Ct method with GAPDH as the normalization control. All the primers for PCR are shown in Additional file 1 : Table S1.
7
Tomato Root Total RNA Extraction and Quantification
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Total RNA was extracted from 100 mg of tomato root using GeneJET Plant RNA Purification Kit (Thermo Scientific™, K0801 from Thermo Fisher Scientific, Waltham, MA, USA) as recommended by the manufacturer. The isolated RNA was DNase-treated with a TURBO DNA-free™ Kit (Invitrogen by Thermo Fisher Scientific), and first-strand cDNA synthesis of 1 µg of total RNA was carried out with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems by Thermo Fisher Scientific), using random hexamers. Real-time PCR was carried out with an ABI 7900 Fast Real-Time System (Applied Biosystems by Thermo Fisher Scientific) with the following protocol: 40 cycles at 95 °C for 15 s, 60 °C for 1 min, using Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific). The relative expression levels were normalized to both the SlEF1 and SlUBI3 reference genes. The normalized relative transcript levels were calculated according to [48 (link),49 (link)] using the 2−ΔΔCt method, where the relative gene expression level of the untreated control was 1. The specific primers for each examined gene are described in Table S1 and related references are cited in the Supplementary Materials .
8
Gene Expression Analysis via qRT-PCR
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Total RNA was isolated from cells with TRIzol reagent (Invitrogen Life Technologies Co, USA) according to manufacturer's protocol. The purity and concentration of total RNA was assessed with a NanoDrop 2000 (Thermo Fisher Scientific, USA). Reverse transcription was accomplished using Prime Script™ RT Reagent Kits with gDNA Eraser (Perfect Real Time, Takara, Japan) with 1 μg of RNA according to the manufacturer's instructions. qRT-PCR was performed with an ABI7900 Fast Real-Time System (Applied Bio systems, USA) using SYBR Premix Ex Taq™ Kits (Takara, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard, and the relative expressions of genes were calculated by the 2-ΔΔCt method [28 (link)]. For each test, three samples were used. Primer sequences are shown in Table 1 .
9
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated from cells with TRIzol reagent (Invitrogen Life Technologies Co, Carlsbad, CA, USA) according to manufacturer’s protocol. The purity and concentration of total RNA was assessed with a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was accomplished using Prime Script™ RT Reagent Kits with gDNA Eraser (Perfect Real Time, Takara, Kusatsu, Japan) with 1 μg of RNA according to the manufacturer’s instructions. qRT-PCR was performed with an ABI7900 Fast Real-Time System (Applied Bio systems, Waltham, MA, USA) using SYBR Premix Ex Taq™ Kits (Takara, Japan). GAPDH was used as an internal standard, and the relative expressions of genes were calculated by the 2−ΔΔCt method [46 (link)]. Primers sequences are shown in Table 1 .
10
Serum miRNA-21 Quantification via qRT-PCR
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The TRIzol™ protocol (Invitrogen, Carlsbad, California, U.S.A.) was used to isolate RNA from the serum samples. Through reverse transcription of RNA using an avian myeloblastosis virus reverse transcriptase (AMV) kit (Takara, Kusatsu, Japan), the extracted complementary deoxyribose nucleic acid (cDNA) was used for polymerase chain reaction (PCR) detection with the 2×SYBR® Green PCR Master Mix (Takara, Kusatsu, Japan). A quantitative real-time PCR (qRT-PCR) system was prepared, using the SYBR® Premix DimerEvaser and including 10.0 μL of deoxynucleoside triphosphate, 0.4 μL of forward primer (10.0 μmol/L), 0.4 μL of reverse primer (10.0 μmol/L), 2.0 μL of cDNA, 0.4 μL of ROX, and 6.8 μL of double-distilled water. The system was subjected to qRT-PCR at 94°C for 5 min before denaturation, followed by 40 cycles at 94°C for 10 s for denaturation, 58°C for 30 s for annealing, and 72°C for an additional 30 s. Relative levels in each sample were determined using the ABI7900 Fast Real-Time System (Applied Biosystems, Massachusetts, U.S.A.) and calculated using the 2−ΔΔCT method and normalized to the internal reference U6. Sequences of miR-21 and U6 were as follows:
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