Levosimendan was purchased from Wako Pure Chemical Industries (Osaka, Japan). Levosimendan was resolved in dimethyl sulfoxide (DMSO) and stored at −80 °C. For GalN/LPS experiments, resolved levosimendan was diluted by 1 mL normal saline for each rat that the concentration of DMSO was decided at 2%. Recombinant human interleukin-1β (IL-1β; 2 × 107 U/mg protein) was purchased from MyBioSource (San Diego, CA, USA). Isoflurane, pentobarbital sodium, collagenase, a Transaminase CII-test kit, GalN, 10% formalin, and a PicaGene Luminescence kit were obtained from Wako Pure Chemical Industries (Osaka, Japan). LPS (Escherichia coli; O111:B4) and mouse anti-β-tubulin were obtained from Sigma–Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits were obtained from Life Technologies (Carlsbad, CA, USA). TRIzol Reagent was obtained from Thermo Scientific (Waltham, MA, USA). T4 polynucleotide kinase, Oligo (dT) Primer, dNTPs Mixture, RNase Inhibitor, and Rever Tra Ace were obtained from Toyobo (Osaka, Japan). Beta-Glo kits and mouse immunoglobulin κ light chain were obtained from Promega (Madison, WI, USA).
Oligo dt primer
Manufactured by Toyobo
Sourced in Japan
Oligo (dT) primers are short synthetic DNA sequences that are complementary to the poly-A tail of mRNA molecules. They are commonly used in reverse transcription reactions to selectively amplify mRNA from a mixed RNA sample.
Automatically generated - may contain errors
Lab products found in correlation
Revertra ace, by Toyobo (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Sybr green pcr master mix, by Roche (4 mentions)
Tri reagent, by Merck Group (3 mentions)
Revertra ace reverse transcriptase, by Toyobo (3 mentions)
Turbo dna free dnase, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Sepasol rna 1, by Nacalai Tesque (2 mentions)
Mmtv reverse transcriptase, by Promega (2 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Nucleospin rna clean up kit, by Macherey-Nagel (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Pentobarbital sodium, by Fujifilm (1 mentions)
Mouse immunoglobulin κ light chain, by Promega (1 mentions)
Isoflurane, by Fujifilm (1 mentions)
Collagenase, by Fujifilm (1 mentions)
Lps escherichia coli o111 b4, by Merck Group (1 mentions)
Mouse anti β tubulin, by Merck Group (1 mentions)
T4 polynucleotide kinase, by Toyobo (1 mentions)
26 protocols using oligo dt primer
1
Levosimendan Inhibits GalN/LPS-induced Liver Injury
2
Quantitative Real-Time PCR Protocol for Gene Expression Analysis
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Total RNA was extracted with TRIZOL Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was synthesized with Superscript II reverse transcriptase using Oligo (dT) primer (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. The PCR amplification was performed for 30 to 35 cycles using an ABI 2720 thermal cycler (Applied Biosystems). PCR System using the following program: 94°C for 5 minutes, 94°C for 30 seconds, annealing at 57-70°C for 30 seconds (Supplementary Table 1 for temperatures used), 72°C for 30 seconds and a final extension at 72°C for 10 minutes. The PCR products were separated on a 1.5% agarose gel, with ethidium bromide staining, and visualized under UV light on a UV transilluminator. GAPDH gene was used as an internal control. Primer sequences are listed in Supplementary Table 1 .
3
RNA Extraction and qPCR Analysis
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RNA was extracted from kidney and liver tissues as well as human renal tubular epithelial cells (HKCs) using a Total RNA Kit (OMEGA, Norcross, GA, USA). Aliquots containing 1 μg of RNA were used for reverse transcription with an oligo-dT primer (Toyobo, Osaka, Japan). qPCR was performed as previously described [18 (link)] using a Roche 480 instrument and SYBR Green PCR Master Mix (Roche, Mannheim, Deutschland) for the genes and corresponding primers (Sangon Biotech, Beijing, China) listed in S1 Table .
4
Quantitative RNA Expression Analysis
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Ribonucleic acid (RNA) was extracted from stomach tissues using a total RNA kit (Omega, Norcross, GA, USA). Aliquots containing 1 μg of RNA were used for reverse transcription with an oligo-dT primer (Toyobo, Osaka, Japan). Real-time quantitative PCR (polymerase chain reaction) (qPCR) was performed as previously described (Ma et al. 2014 (link)) using a Roche 480 instrument and SYBR green PCR master mix (Roche, Mannheim, Germany). The primers (Sangon biotech, Beijing, China) were listed in Table 1 . Expression levels were calculated using the 2–△△Ct method, with the Ct values normalised using GAPDH as an internal control.
5
Quantifying ESR1 mRNA Expression in MCF7 Cells
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Total RNA was isolated from MCF7 cells transfected with the indicated vectors using Fast Gene RNA Basic kit (Nippon Genetics, Tokyo, Japan). One hundred nanograms of total RNA were added to a mixture of 250 nM Oligo dT primer (Toyobo), 500 µM dNTPs, 40 U RNase inhibitor (Takara Bio. Inc.) and 10 U M-MuLV reverse transcriptase (NewEngland Biolabs), and incubated at 42 °C for 60 min and 90 °C for 10 min. The expressions of ESR1 mRNA were measured using the synthesized DNAs as templates by a Quanti Tect SYBR Green PCR kit (Qiagen) with a Rotor-gene 6000 (Qiagen) at 95 °C for 15 min, followed by 45 cycles of 94 °C for 15 sec, 55 °C for 30 sec and 72 °C for 30 sec. The primers used for qPCR are shown in Supplementary Table 10S . The calculated data were normalized to the values for GAPDH used as a control gene.
6
Quantitative Real-Time PCR Analysis
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Total hepatic RNA was prepared by a total RNA kit (OMEGA, Georgia, USA) in accordance with instructions. An aliquot of 1 μg RNA was applied for reverse transcription with oligo-dT primer (TOYOBO, OSAKA, Japan). Quantitative real-time PCR was performed using the Roche 480 instrument (Roche, Mannheim, Germany) and SYBR Green PCR Master Mix (Roche, Mannheim, Germany) for the subsequent genes with the corresponding primers (Sangon Biotech, Beijing, China) (Supplementary Table S6 ). Quantification was conducted using the ΔΔCT method [24 (link)]. The quantity of messenger RNA was normalized with the internal standard GAPDH.
7
Quantitative Gene Expression Analysis in Mouse Heart
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RNA was extracted from heart tissues with the Total RNA Kit (OMEGA, Norcross, GA, USA). cDNA synthesis was performed with an oligo-dT primer (TOYOBO, Osaka, Japan) according to the manufacturer's instructions in a reaction volume of 20 μl. For quantitative mRNA analysis, a template equivalent to 20 ng of RNA was subjected to 40 cycles of quantitative real-time polymerase chain reaction (qPCR) using a CFX real-time PCR instrument (Bio-Rad, Hercules, CA, USA) with SYBR Green PCR Master Mix (TransGen, Beijing, China) in triplicate. GAPDH was used for normalization. Relative mRNA expression was calculated using the 2−ΔΔCt method. The primer sequences (forward and reverse) of mouse Wip1, collagen I, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and GAPDH were as follows:
Wip1 (forward 5’-CTGACTGATAGCCCTACTTAC AACA-3’ and reverse 5’-GAGAAGGCATTACTGC GAACA-3’);
Collagen I (forward 5’-GAGCGGAGAGTACTGGATCG-3’ and reverse 5’-TACTCGAACGGGAATCCATC-3’);
IL-6 (forward 5’-TCCAGTTGCCTTCTTGGGACTGAT-3’ and reverse 5’-TAAGCCTCCGACTTGTGAAGTGGT-3’);
TNF-α (forward 5’-TTCCGAATTCACTGG AGCCTCGAA-3’ and reverse 5’-TGCACCTCAGGGAAGA ATCTGGAA-3’);
IL-1β (forward 5’-TGGAGAGTGTGGATCC CAAGCAAT-3’ and reverse 5’-TGCTTGTGAGGTGCTGAT GTACCA-3’); and
GAPDH (forward 5’-AACTTTGGCATTGTGGAAGG-3’ and reverse 5’-ACACATTGGGGGTAGGAACA-3’).
Wip1 (forward 5’-CTGACTGATAGCCCTACTTAC AACA-3’ and reverse 5’-GAGAAGGCATTACTGC GAACA-3’);
Collagen I (forward 5’-GAGCGGAGAGTACTGGATCG-3’ and reverse 5’-TACTCGAACGGGAATCCATC-3’);
IL-6 (forward 5’-TCCAGTTGCCTTCTTGGGACTGAT-3’ and reverse 5’-TAAGCCTCCGACTTGTGAAGTGGT-3’);
TNF-α (forward 5’-TTCCGAATTCACTGG AGCCTCGAA-3’ and reverse 5’-TGCACCTCAGGGAAGA ATCTGGAA-3’);
IL-1β (forward 5’-TGGAGAGTGTGGATCC CAAGCAAT-3’ and reverse 5’-TGCTTGTGAGGTGCTGAT GTACCA-3’); and
GAPDH (forward 5’-AACTTTGGCATTGTGGAAGG-3’ and reverse 5’-ACACATTGGGGGTAGGAACA-3’).
8
Quantitative RT-PCR Analysis of Gene Expression
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Total hepatic RNA was extracted by using a total RNA kit (OMEGA, Georgia, U.S.A) according to manufacturer instructions. An aliquot of 1 μg RNA was applied for reverse transcription with oligo-dT primer (TOYOBO, OSAKA, Japan). Quantitative real-time PCR was performed using the Roche 480 instrument (Roche, Mannheim, Germany) and SYBR Green PCR Master Mix (Roche, Mannheim, Germany) for the subsequent genes with the corresponding primers (Sangon Biotech, Beijing, China) (Supplementary Table S5 ). Quantification was performed by the ΔΔCT method. The quantity of mRNA was normalized with the internal standard GAPDH.
9
Comparative Toxicology Assay Protocol
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All of the test reagents used were of reagent grade. TRI reagent, Dulbecco's Modified Eagle's Medium (DMEM), β-carotene, and goat anti-rabbit IgG were purchased from Sigma Chemical Co. (St. Louis, MO). Oligo(dT) primer, ReverTra Ace, and reverse transcriptase (RT) buffer were purchased from Toyobo (Osaka, Japan). Polyclonal rabbit anti-human AhR antibody was purchased from Abcam (Tokyo, Japan). Primer sets were purchased from Invitrogen (Carlsbad, CA). Lead acetate, As. Acid, and all other reagents were of analytical grade and purchased from Wako Pure Chemical Industries (Tokyo, Japan).
10
Reagent Procurement for Molecular Analysis
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All of the test reagents used were of reagent grade, including those described below.
TRI reagent was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Oligo(dT) primer, reverse transcriptase buffer, and ReverTra Ace were purchased from Toyobo (Osaka, Japan).
The S9 cofactor was purchased from Oriental Yeast (Tokyo, Japan). All other reagents were of analytical grade or of the highest quality available; they were purchased from Wako Pure Chemical Industries (Tokyo, Japan).
TRI reagent was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Oligo(dT) primer, reverse transcriptase buffer, and ReverTra Ace were purchased from Toyobo (Osaka, Japan).
The S9 cofactor was purchased from Oriental Yeast (Tokyo, Japan). All other reagents were of analytical grade or of the highest quality available; they were purchased from Wako Pure Chemical Industries (Tokyo, Japan).
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