Organic solvents were purchased from J.T. Baker (Ulsan, Korea). Dulbecco’s modified Eagle’s medium (DMEM), DMEM with F-12 Ham 1:1 mixture (DMEM/F-12), fetal bovine serum (FBS), and horse serum were procured from Hyclone (South Logan, UT, USA). Hydrogen peroxide (H2O2), ascorbic acid, dimethyl sulfoxide (DMSO), dichlorofluorescein acetate (DCF-DA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were purchased from Sigma-Aldrich (St. Luis, MO, USA). RNA-Bee reagent and GoTaq®® Green Master Mix were purchased from AMS Bio (Abingdon, UK) and Promega (Madison, WI, USA), respectively. All the primary antibodies that recognized glutathione S-transferase (GST) m5, glutathione peroxidase (GPX) 4, peroxiredoxin (PRX) 3, phosphorylated cAMP response element-binding protein (P-CREB), nectin-2 and β-actin were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).
Rna bee reagent
Manufactured by AMS Biotechnology
Sourced in United Kingdom, United States
RNA-Bee is a reagent designed for the isolation and purification of total RNA from a variety of biological samples. It is a guanidinium thiocyanate-phenol-based solution that effectively lyses cells and denatures RNases, allowing for the efficient extraction of high-quality RNA.
Automatically generated - may contain errors
Lab products found in correlation
Gotaq green master mix, by Promega (2 mentions)
Iscript, by Bio-Rad (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Faststart universal sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
P creb, by Santa Cruz Biotechnology (1 mentions)
M mlv rt, by Thermo Fisher Scientific (1 mentions)
Spectramax quickdrop, by Molecular Devices (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rnase h, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Promega (1 mentions)
11 protocols using rna bee reagent
1
Antioxidant and Neuroprotective Assays
2
Apigenin Modulates Oxidative Stress Response
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GC-2spd cells were plated at 2 × 105 cells/well and incubated for 24 h. After incubation period, the cells were cultured in the presence of apigenin at various concentration for 2 h, and then stimulated by H2O2 for 1 h. Total RNA was extracted from the cells using RNA-Bee reagent according to the manufacturer’s instruction (AMS Bio, Abingdon, UK) and quantified using SpectraMax QuickDrop (Molecular Devices, San Jose, CA, USA). RNA (1 μg) was reverse-transcribed for 50 min at 37 °C in the mixture containing 1 μL oligo (dT), 10 mM dNTP, 0.1 M DTT, 5X PCR buffer and 1μL M-MLV RT (Invitrogen Co., Carlsbad, CA, USA). An aliquot (200 ng) of RT products was amplified in a 25 μL reaction by a GoTaq® Green Master Mix (Promega Co., Madison, WI, USA) in the present of 10 pM oligonucleotide primer. The primers used for RT products from the cells was shown in Table S1 . The PCR was performed for 30 cycles at 95 °C for 30 s, 56 °C for 40 s, and 72 °C for 40 s. After amplification, PCR products were separated on 2% agarose gel containing ethidium bromide (Et-Br) by electrophoresis, and the bands were visualized by ultraviolet fluorescence. The experiment was performed in triplicate. The intensities of the bands were analyzed using NIH Image J 1.4 package and normalized to GAPDH value.
3
Quantitative RT-PCR for Gene Expression
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Total RNA was isolated from tissue and cells using RNA-Bee reagent (Amsbio) and was reverse transcribed into cDNA using iSCRIPT (BioRad). Reactions were performed on the LightCycler 480 II (Roche) using 0.4 uM primers and Faststart Universal SYBR Green PCR Master Mix (Applied Biosystems). Primer sequences are described in Supplementary Table S1 .
4
RNA Isolation and RT-qPCR Analysis
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RNA was isolated using RNA Bee reagent (Amsbio) or Trizol LS reagent (Life Technologies) according to the manufacturer’s instructions. All RNA samples were treated with DNase I using RNeasy Micro Kit (Qiagen) and reverse-transcribed into complementary DNA using Super-RT enzyme (HT Biotechnology). RT-PCR was performed using KOD Hot Start DNA polymerase (Novagen) and self-designed primers (Table S1 ). Products were run on a 2% agarose gel and visualised using a Bio-Rad ChemiDoc XRS + System and Image Lab 5 software. Real-time qPCR was performed using SYBR Green mastermix (Applied Biosystems) and self-designed primers or Taqman Universal PCR master mix (Applied Biosystems) and commercially available Taqman gene expression assays (Table S1 , Applied Biosystems). Samples were run on the ViiA7 real-time PCR system (Applied Biosystems).
5
RNA Extraction and Reverse Transcription
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This method has been described elsewhere.24 Briefly, total RNA extraction was performed using the RNA Bee reagent (Amsbio, Abingdon, UK) according to the manufacturer's instructions. Twenty per cent of the RNA obtained from a whole cell‐sort sample was denatured at 96° for 3 min with 0·5 μg oligo‐dT and placed on ice before reverse transcription in a 50‐μl reaction for 1 hr at 42° with 1× RT buffer, 0·2 mm dNTPs, 1 mm dithiothreitol, 800 Uml‐1 RNAsin (Promega, Southampton, UK) and 200 U Superscript II reverse transcriptase. After heat inactivation, reactions were digested for 20 min with 2 U RNase H (all Thermo Fisher, Paisley, UK except where indicated).
First‐round PCR was done with 2 μl of cDNA reaction with standard reaction conditions using the manufacturer‐supplied buffer with Hotstart Pfu turbo polymerase (Agilent Stratagene, Stockport, UK) and 25 pmol of the appropriate primer in a 50‐μl reaction for 34 cycles with the following cycling: 96°, 30 seconds; 59°, 30 seconds; 72°, 2 min; preceded by an incubation at 96° for 3 min. All primers used are described elsewhere.24
First‐round PCR was done with 2 μl of cDNA reaction with standard reaction conditions using the manufacturer‐supplied buffer with Hotstart Pfu turbo polymerase (Agilent Stratagene, Stockport, UK) and 25 pmol of the appropriate primer in a 50‐μl reaction for 34 cycles with the following cycling: 96°, 30 seconds; 59°, 30 seconds; 72°, 2 min; preceded by an incubation at 96° for 3 min. All primers used are described elsewhere.
6
RNA Extraction and Quantification Protocol
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RNABee reagent (AMS Bio, Abingdon, UK) was used to extract the total RNA according to the manufacturer's instructions, and 1 μg of extracted RNA was reverse-transcribed as previously described [28 (link)]. Polymerase chain reaction (PCR) was performed as described in our previous report [14 (link)], and the primers used for the study are shown in Table 1 . Band intensities were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band analyzed by NIH ImageJ software (version 1.41o; National Institutes of Health, Bethesda, MD, USA).
7
RNA Extraction and qRT-PCR Analysis
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RNABee reagent (AMS Bio, Abingdon, UK) was used to extract the total RNA according to the manufacturer’s instructions, and 1 µg of extracted RNA was reverse-transcribed as previously described [28 (link)]. Polymerase chain reaction (PCR) was performed as described in our previous report [27 (link)], and the primers used for the study are shown in Table 1 . Band intensities were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band analyzed by NIH ImageJ software (version 1.41o; National Institutes of Health, Bethesda, MD).
8
RNA Extraction and qRT-PCR Quantification
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RNA-Bee reagent (AMS Bio, Abingdon, UK) was used to extract the total RNA according to the manufacturer’s instructions. RNA (1 µg) was reverse transcribed for 50 min at 37 °C in a mixture containing 1 µL oligo (dT), 10 mM dNTP, 0.1 M dithiothreitol, 5 X polymerase chain reaction (PCR) buffer, and 1 µL Moloney Murine Leukaemia Virus Reverse Transcriptase (RT) (Invitrogen Co., Carlsbad, CA, USA). An aliquot (200 ng) of the RT products was amplified in a 25 µL reaction volume using a GoTaq® Green Master Mix (Promega Co., Madison, WI, USA) in the presence of 10 pM oligonucleotide primer. The primers used for the RT products was shown in Table 1 . The PCR was performed as described in our previous report (Kopalli et al. 2016 (link)). The intensity of the bands was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analysed using the ImageJ software package (version 1.41o; National Institutes of Health, Bethesda, MA, USA).
9
Quantitative RT-PCR for Gene Expression
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Total RNA was isolated from tissue and cells using RNA-Bee reagent (Amsbio) and was reverse transcribed into cDNA using iSCRIPT (BioRad). Reactions were performed on the LightCycler 480 II (Roche) using 0.4 uM primers and Faststart Universal SYBR Green PCR Master Mix (Applied Biosystems). Primer sequences are described in Supplementary Table S1 .
10
qRT-PCR Analysis of Gene Expression
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Cells were stimulated with pLTA and/or aLTA for the indicated time and total RNAs were extracted using RNA-Bee reagent (AMS Biotechnology, Cambridge, MA, USA). Total RNA (1.0 μg) was used for cDNA synthesis (iScript cDNA Synthesis kit; Bio-Rad, Hercules, CA, USA). The expression level of messenger RNA (mRNA) was measured by real-time PCR using the CFX Connect™ Real-Time PCR detection system (Bio-Rad), and the PCR products were detected with SYBRⓇ Premix Ex II (TaKaRa, Japan). The sequences for the forward and reverse primer pairs are listed in Supplement Table S1 . The comparative Δ−ΔCt method was carried out as outlined by Livak and Schmittgen [11 (link)]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the detected gene expression and fold change of experimental samples was estimated when untreated or control samples were set to 1.
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