A. pasteurianus strains were inoculated in LYSE medium at 30 °C with vigorous agitation for 12 h. RNA was extracted using SV Total RNA Isolation System (Promega, Madison, WI, USA), and then reverse-transcribed using PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China) and four specific primers alsDdw1, alsS1dw1, alsS2dw1, and gyrAdw1. Real-time PCR assays were performed in triplicates on the Applied Biosystems ABI 7500 Real-time PCR System using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Dalian, China). The relative transcriptional levels of target genes were calculated using the 2−ΔΔCt method, and the gyrA gene was used as the reference gene.
Abi 7500 real time pcr system
Manufactured by Takara Bio
Sourced in Japan, China, United States
The ABI 7500 Real-Time PCR system is a thermal cycler designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of detecting and quantifying target DNA sequences in real-time during the amplification process. The system utilizes fluorescent dyes or probes to monitor the accumulation of PCR products throughout the reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (23 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (7 mentions)
Primescript rt reagent kit, by Takara Bio (6 mentions)
Sybr premix ex taq, by Takara Bio (5 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (5 mentions)
Sybr green, by Takara Bio (4 mentions)
Sybr green pcr master mix, by Takara Bio (3 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Sybr green master mix, by Takara Bio (2 mentions)
Primescript rt master mix kit, by Takara Bio (2 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Primescript rt kit, by Takara Bio (2 mentions)
Sybr premix ex taqtm 2 kit, by Takara Bio (2 mentions)
Sybr green pcr kit, by Takara Bio (2 mentions)
Reverse transcription system, by Promega (2 mentions)
Rnaiso plus kit, by Takara Bio (2 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
Sv total rna isolation system, by Promega (1 mentions)
53 protocols using abi 7500 real time pcr system
1
Transcriptional Analysis of A. pasteurianus
2
Transcriptional Profiling of IbSSI in Sweet Potato
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The transcript levels of IbSSI were analyzed in five different tissues (storage root, fibrous root, stem, leaf and petiole) of Xu 781 grown for 100 days in a field. Total RNA was extracted from each tissue and reverse transcribed into cDNA and quantitative real-time PCR (qRT-PCR) was conducted using the SYBR detection protocol (TaKaRa) with the ABI 7500 Real-Time PCR system. The primers used to amplify IbSSI and IbActin (endogenous control) are listed in Supplementary Table S1 .
The response of IbSSI to exogenous sucrose was investigated as described by Wang et al.42 (link) with some modifications. Leaf-petioles (10 cm) from Xu 781 grown for 100 days in a field were cultured for 1 day in water in the dark and then treated with either water (control) or 175 mM sucrose in the dark at 28 °C. qRT-PCR was conducted to determine the mRNA levels of IbSSI in cuttings harvested at different time points (0, 2, 4, 6, 12, 24 and 48 h) after treatment.
The response of IbSSI to changes of light conditions was examined as described by Leterrier et al.62 (link). Twelve in vitro-grown plants of Xu 781 were acclimated to a 16-h-light/8-h-dark regimen in a growth chamber at 28 °C for 1 month prior to modifying the light conditions. Total RNA was extracted from each whole plant sampled at different time points (shown in Fig.2C ) and the mRNA levels of IbSSI were subsequently detected by qRT-PCR.
The response of IbSSI to exogenous sucrose was investigated as described by Wang et al.42 (link) with some modifications. Leaf-petioles (10 cm) from Xu 781 grown for 100 days in a field were cultured for 1 day in water in the dark and then treated with either water (control) or 175 mM sucrose in the dark at 28 °C. qRT-PCR was conducted to determine the mRNA levels of IbSSI in cuttings harvested at different time points (0, 2, 4, 6, 12, 24 and 48 h) after treatment.
The response of IbSSI to changes of light conditions was examined as described by Leterrier et al.62 (link). Twelve in vitro-grown plants of Xu 781 were acclimated to a 16-h-light/8-h-dark regimen in a growth chamber at 28 °C for 1 month prior to modifying the light conditions. Total RNA was extracted from each whole plant sampled at different time points (shown in Fig.
3
Quantitative Real-Time PCR Analysis
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Total RNA of transfected cells was prepared, and 2 μg of total RNA was reverse transcribed into cDNA using the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara) according to the manufacturer’s protocol. Quantitative real-time PCR was performed in triplicate on an ABI 7500 Real-Time PCR system using SYBR Green Master Mix (Takara). The mRNA levels were normalized to the expression of the housekeeping gene β-actin.
4
Quantification of ER Stress Markers in HepG2 Cells
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The RNA extraction obtained from HepG2 cells was accomplished using 1 mL of Trizol (Beijing Lan Y Science & Technology) per well in 6-well plates. Reverse transcription was performed using the PrimeScriptTMRT Master Mix kit (TaKaRa, Otsu, Japan); real-time PCR was conducted using the TB Green® Premix Ex TaqTM Ⅱ kit (TaKaRa, Otsu, Japan) and tested with the ABI 7500 Real-Time PCR System. The mRNA expressions of the target genes included CHOP, 78 kDa glucose-regulated protein (GRP78), activating transcription factor 6 (ATF6), protein disulfide isomerase family A member 6 (PDIA6), BAX, BCL2, and CASP3. Primers were designed with Primer Premier 6 (Premier Biosoft, CA, USA) and the primer sequences are illustrated in Table 2 . The results were normalized to the β-actin gene expression, and the mRNA expression was calibrated with the CON value. Fluorescence results were calculated in relation to the β-actin CT value using the 2ΔΔCT method [41 (link),42 (link),43 (link)].
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from tissues and cells using RNAiso Plus (TaKaRa, China) and then cDNA was synthesized using a PrimeScriptTM RT reagent Kit (TaKaRa), according to the manufacturer’s instructions. mRNA expression levels were assessed by qRT-PCR using a SYBR Premix Ex Taq Kit (TaKaRa) on an ABI 7500 Real-Time PCR System, with GAPDH used as internal control. Primers used in qRT-PCR are listed in (Supplementary Table S1 ).
6
Quantitative analysis of circular RNA circ_0010729 and its target genes
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Total RNA was extracted using Trizol reagent (Invitrogen) from cells. Then reverse transcription was performed using 1 μg of total RNA with a reverse transcription kit (Takara, Tokyo, Japan) to synthesize cDNA. Subsequently, qRT-PCR was conducted using SYBR Green PCR master mix (Takara) on an ABI 7500 Real-Time PCR system. The 2−ΔΔCt method was used to calculate the fold changes with U6 or glyceraldehyde 3-phosphate dehydrogenase (GADPH) as an internal control. The same experiment was repeated three times, and the average was taken. The following primers were used: circ_0010729: F, 5'-CAGGCAGAGGTCCGGGCCTGTT-3' and R, 5'-GGACCGTTCTCAATGGCGTATAC-3'; GADPH: F, 5'-GGTGAAGGTCGGAGTCAAC-3' and R, 5'-AGAGTTAAAAGCAGCCCTGGTG-3'; TRAF6: F, 5'-CAGTGGTCGTATCGTGCTTA-3' and R, 5'-CCTTATGGT TTCTTGGAGTC-3'; miR-370-3p: F, 5'-GCCTGCTGGGGTGGAACCTGGT-3' and R, 5'-CTCAACTGGTGTCGTGGA -3'; U6: F, 5'-CTCGCTTCGGCAGCACA-3' and R, 5'-AACGCTTCACGAATTTGCGT-3'.
7
Quantifying IL-10 mRNA Expression in Clinical Samples
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Total RNA was extracted from the clinical tissue samples using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized using the PrimeScript RT Master Mix kit (Takara Biotechnology Co., Ltd.). Subsequently, qPCR was performed to examine the expression of IL-10 using SYBR premix Ex Taq (Takara Biotechnology Co., Ltd.) on an ABI 7500 Real-Time PCR System. Relative mRNA expression levels were calculated using the 2−∆∆Cq method (15 (link)), with GAPDH as the internal control. The thermocycling conditions were 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The primers were as follows: IL-10, 5′-GCCAGAGCCACATGCTCCTA-3′ (forward) and 5′-GATAAGGCTTGGCAACCCAAGTAA-3′ (reverse); and GAPDH, 5′-AAATGGTGAAGGTCGGTGTGAAC-3′ (forward) and 5′-CAACAATCTCCACTTTGCCACTG-3′ (reverse).
8
Quantifying Gene Expression in Mouse Ileum
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Total RNA was isolated from ileum tissue using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA). cDNA was synthesized from 1 μg of total RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time quantitative polymerase chain reaction (qPCR) was performed using the ABI 7500 Real-Time PCR system and SYBR Premix Ex Taq™ II Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. Oligonucleotide primers for mouse zonula occludens- (ZO-) 1 (forward 5′-ACTCCCACTTCCCCAAAAAC-3′ and reverse 5′-CCACAGCTGAAGGACTCACA-3′) and β-actin (forward 5′-ACAGGCATTGTGATGGACTC-3′ and reverse 5′-ATTTCCCTCTCAGCTGTGGT-3′) were synthesized by Sangon Biotech.
9
MALAT1 Expression Quantification
10
RNA Extraction and Gene Expression Analysis
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Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RT-PCR was performed on 0.5 mg total RNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). qPCR was performed using an ABI 7500 Real-Time PCR system and a SYBR Premix Ex TaqTM II kit (Takara). Gene expression of CHD5 was normalized to β-actin and all samples were analyzed in triplicate. Primers for qRT-PCR are shown in Table 1 .
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