Total RNA from disc cells was extracted using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. For cDNA synthesis, 2 µg total RNA was reverse transcribed in a reaction mixture containing 25 units of ribonuclease inhibitor, 15 units of reverse transcriptase, 500 ng Oligo (dT) primers, 3 mM MgCl2, 0.5 mm dNTP, and 1× RT buffer (Promega). Synthesized cDNA was used as a template for polymerase chain reaction (PCR) amplification with GoTaq polymerase (Promega) and primers for Fas (Bioneer, Cheongwon, Korea). PCR was performed with the Mycycler thermal cycler (BioRad, Hercules, CA, USA) according to the manufacturer's instructions. Analysis of the PCR products in 1% agarose gels showed single-band amplification products of expected sizes. Primer sequences and experimental conditions of PCR are summarized in Tables 1 and 2 . Blots were semiquantitatively analyzed using GF670 imaging densitometer and the Molecular Analyst software (Bio-Rad), and results were expressed relative to the expression of Gapdh housekeeping gene. All experiments were repeated three times per sample, and average of three technical replicates was used as the final band density.
Rt buffer
Manufactured by Promega
Sourced in United States
RT buffer is a buffer solution used in reverse transcription (RT) reactions, which are a crucial step in the process of converting RNA into complementary DNA (cDNA). The core function of the RT buffer is to provide the necessary conditions, such as pH and ionic strength, for the reverse transcriptase enzyme to efficiently and accurately convert RNA into cDNA, which can then be used for various downstream applications, such as gene expression analysis and detection of specific RNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Gotaq polymerase, by Promega (5 mentions)
M mlv reverse transcriptase, by Promega (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Mycycler thermal cycler, by Bio-Rad (3 mentions)
Rnase inhibitor, by Promega (3 mentions)
Dnase, by Thermo Fisher Scientific (3 mentions)
Trizol regent, by Thermo Fisher Scientific (2 mentions)
Dntps, by Bioneer (2 mentions)
Tri reagent, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Amv reverse transcriptase, by Promega (2 mentions)
Rnasin, by Promega (2 mentions)
Takara premix taq polymerase, by Takara Bio (2 mentions)
Biomark hd system, by Standard BioTools (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Molecular analyst software, by Bio-Rad (1 mentions)
Oligo dt primer, by Promega (1 mentions)
Hexanucleotide mix, by Roche (1 mentions)
Quantitect primer assay, by Qiagen (1 mentions)
M mlv rt, by Promega (1 mentions)
19 protocols using rt buffer
1
Fas Gene Expression Analysis
2
Quantitative Gene Expression Analysis
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Total RNA was isolated from cell lysates using RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen). First, cDNA synthesis was performed using 80 ng RNA with RT Buffer (Promega), dNTPs (GE Healthcare), Hexanucleotide Mix (Roche) and MMLV RT (Promega). For the preamplification step PreAmp Master Mix, SuperScript III First-Strand Synthesis SuperMix (both Thermo Fisher), TE buffer (Ambion) was used according to the manual. To quantify the amount of the genes of interest QuantiTect Primer Assays (Qiagen) were used on the BioMark HD System with 96.96 Dynamic Arrays (both Fluidigm). Relative gene expression was calculated as a ratio of target gene concentration to the housekeeping gene concentration. Details have been described in38 ,40 (link).
3
RNA Extraction and RT-PCR Analysis
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Total RNA from BV2 cells and mouse brain tissue was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). 1 μg of RNA was reverse transcribed in a reaction mixture containing 1 U RNase inhibitor, 500 ng random primer, 3 mM MgCl2, 0.5 mM dNTP, 1X RT buffer, and 10 U reverse transcriptase (Promega, Madison, WI, USA) for RT-PCR. The cDNA was used as a template for the PCR reaction, which was carried out with Go Taq polymerase (Promega) and primers. The RT-PCR was performed on a Bio-Rad T100 thermal cycler (Bio-Rad, Richmond, CA, USA). The ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used to perform quantitative RT-PCR using Sensi FASTTM SYBR Hi-ROX Mix (Bioline, London, UK). The following formula was used to normalize the expression levels of the target genes towards GADPH: 2 (Ct, test gene-Ct, GAPDH). Table 1 lists the primer sequences utilized in the PCR experiments.
4
Quantifying Gene Expression in BV2 Cells and Brain Tissue
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BV2 cells (7.5×105 cells per well in a 6-well plate) were treated with poly(I:C) in the presence or absence of galangin, and total RNA was extracted using TRI reagent (Thermo Fisher Scientific). Cortical brain tissue was homogenized using a homogenizer (Fisher Scientific, Pittsburgh, PA, USA), and total RNA was extracted using TRI reagent. For RT-PCR, total RNA (1 μg) was reverse-transcribed in a reaction mixture containing 0.1 μg of random primers, 3 mM MgCl2, 0.5 mM dNTP, 1× RT buffer, and 10 U reverse transcriptase (Promega). The synthesized cDNA was used as a template for the PCR reaction using Go Taq polymerase (Promega) and primers for the target gene. The primers used in the PCR reaction are shown in Table 1 .
5
RNA Extraction and qPCR Analysis
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Total RNA was prepared from cells with TRIzol reagent (Thermo Scientific, Seoul, Korea) according to the manufacturer’s instructions. Total RNA (1 μg) and 50 μM oligo-dT primers were mixed in 17 μL of DEPC-treated water and reacted at 70 °C for 5 min. After the reaction, 2 μL of 10 mM dNTP (Takara Bio Inc., Seoul, Korea), 5 μL of 5 × RT buffer (Promega, Seoul, Korea), and 1 μL of 200 unit/μL M-MLV RTase (Promega, Seoul, Korea) were added and cDNA was synthesized by reaction at 25 °C for 5 min, at 42 °C for 60 min and at 70 °C for 15 min. Quantitative real-time PCR (qPCR) was performed using a RotorGene 6000 (Qiagen, Seoul, Korea) with reaction mixtures containing 2 × SensiFast SYBR NO-ROX kit (Bioline, Seoul, Korea), 100 ng of cDNA, and 10 pM of each primer (Table 1 ). After amplification, melting curve analysis was performed to verify the specificity of the reactions. The end point used in the real-time PCR quantification, Ct, was defined as the PCR threshold cycle number. The relative quantification of the target gene expression was evaluated using the ΔΔCt method [60 ]. The fold change of the target gene expression relative to the control (GAPDH) was calculated as the 2−ΔΔCt value, which was arbitrarily defined as 1.
6
RT-PCR Assay for Fas Gene Expression
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The RT-PCR experimental method used in this study was the same as described in a previous paper [10 (link)]. Briefly, total RNA was extracted from NP cells and tissues using TRIzol regent (Invitrogen, Grand Island, NY, USA) following the manufacturer’s instructions. For complementary DNA (cDNA) synthesis, 2 μg total RNA was reverse transcribed in a reaction mixture containing 25 units ribonuclease inhibitor, 15 units reverse transcriptase, 500 ng oligo (dT) primer, 3 mM MgCl2, 0.5 mm dNTP, and 1× RT buffer (Promega Corp., Madison, WI, USA). cDNA was used as a template for PCR using GoTaq polymerase (Promega Corp.) and PCR primers to amplify Fas and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioneer, Cheongwon, Korea). PCR was performed using a Mycycler thermal cycler (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. An analysis of the PCR products using 1% agarose gel electrophoresis revealed single amplicons of the expected sizes. GAPDH was used as an internal control. The primer sequences used in the experiment are as follows: 5-GGCATCTGGACCCTCCTACCTCTG-3 (forward) and 5-CCTTGGAGTTGATGTCAGTCACTTGG-3 (reverse) for Fas and 5-ATCATCTCCGCCCCTTCTGC-3 (forward) and e 5-GCCTGCTTCACCACCTTCTT-3 (revers) for GAPDH.
7
Quantification of COX-2 and NDRG2 Expression
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Total RNA from the harvested cells was isolated using the TRIzol reagent (Invitrogen), and it was reverse transcribed into cDNA using an oligo-(dT) primer, dNTPs (Bioneer, Korea), MMLV reverse transcriptase, and RT buffer (Promega, USA). The cDNAs were amplified by PCR in a reaction mixture containing cDNA, each specific primer, dNTPs, and Top DNA polymerase. The primers and reagents were purchased from Bioneer. The PCR products were electrophoresed in 1% agarose gels containing ethidium bromide. The following primers were used: 5′-CTGAATGGGGTGATGAGCAG-3′ and 5′-ATTCCTACCACCAG CAACCC-3′ for COX-2; 5′-GGATTCATGGCGGAGCTGCAGGA GG-3′ and 5′-GAATTCTCAACAGGAGACCTCCATGGT-3′ for NDRG2; and 5′-CCACACCTTCTACAATGAGC-3′ and 5′-TGA GGTAGTCAGTCAGGTCC-3′ for β-actin. For the quantitative real-time PCR, the amplification was performed using an ABI StepOnePlus™ Real-Time PCR thermal cycler with Power SYBR Green PCR Master Mix according to the manufacturer’s protocol (Applied Biosystems, USA). Target mRNA levels were normalized against GAPDH, and the relative mRNA expression levels were calculated. The experiments were performed in triplicate. The following primers were used: 5′-AGGGTTGCTGGTGG TAGGAA-3′ and 5′-GGTCAATGGAAGCCTGTGATACT-3′ for COX-2; and 5′-CTGGGCTACACTGAGCACCAG-3′ and 5′-CCA GCGTCAAAGGTGGAG-3’ for GAPDH.
8
Quantitative Analysis of tRNA Methylation
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Small RNAs extracted from wild-type and METTL8 KO cell lines, in vitro methylated with recombinant His14-MBP-METTL8/METTL8D230A/METTL8D309A or untreated, were pre-annealed to 0.125 pmol (for mt-tRNASer(UCN)) or 0.250 pmol (for mt-tRNAThr) of a 5′-[32P]-radiolabelled DNA oligonucleotide (Supplementary Table 6 ) in 1x First Strand buffer (ThermoFisher Scientific) by heating at 95 °C for 3 min and allowing the mixture to cool slowly to 55 °C. Extension mix (10 U/μL SuperScript III (ThermoFisher Scientific), 1 U/μl Ribolock (ThermoFisher Scientific) and 0.08 mM dNTPs in 1× First Strand buffer) was added to each reaction and samples were incubated at 55 °C for 30 min. Reactions were stopped by adding 2× formamide dye (95% formamide, 0.5 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol, 0.025% SDS) and samples were heated at 85 °C for 5 min. Samples were resolved in 15% denaturing (7 M urea) polyacrylamide gels, which were dried and exposed to a phosphorimager screen (cytiva) overnight. Radiolabelled cDNA fragments were detected using a phosphorimager Typhoon FLA 9500 (cytiva) and quantified using the Image Studio 5.2.5 software (LI-COR). For primer extension analyses of RNAs derived from cells treated with siRNAs against OSGEPL1 or TRIT1, 2 U/μL AMV reverse transcriptase (Promega) was used in 1× RT buffer (Promega) and extension was performed at 58 °C for 1 h.
9
Comprehensive RNA Isolation and cDNA Synthesis
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Total RNA was isolated from the nine individuals sampled per cross per time point (145, 234, and 327 days post-fertilization) using RNeasy kits (QIAGEN, Valencia, CA, USA) following the manufacture’s recommendations with in-column DNase treatment. RNA was eluted in 50 μl of RNase-free water, quantified, and stored at -80°C. cDNA synthesis was performed using 1 μg of RNA with 1X RQ1 Buffer (Promega, Madison, WI, USA), 1 unit RNasin (Promega), 0.25 units DNase I (Promega), and incubation at 37°C for 30 minutes followed by 70°C for 5 minutes. Next, 125 ng each of oligo (dt)15 and random hexamer primers (Promega) were added and annealed at 70°C for 5 minutes, followed by 2 minutes on ice. Finally, 1mM dNTP (Promega), 1.25X RT buffer (Promega), 1 unit RNasin (Promega), and 100 units of M-MLV reverse transcriptase (Promega) were added and incubated at 42°C for 60 minutes, followed by 70°C for 15 minutes. All cDNA was stored at -20°C.
10
Total RNA Isolation and qRT-PCR Analysis
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Total RNA from harvested cells was isolated using the TRI reagent (Molecular Research Center Inc, Cincinnati, OH, USA). To synthesize cDNA, 5 μg of total RNA was first incubated with an oligo-dT primer at 70°C to denature the secondary structure of the RNA and then incubated at room temperature for 10 min to allow the annealing of the primer. Then, dNTPs (Bioneer, Daejeon, Korea), M-MLV reverse transcriptase (Promega, Madison, WI, USA) and RT buffer (Promega) were added to the reaction. The mixture was incubated for 3 h at 37°C and then boiled at 100°C for 3 min. β-actin sequences were amplified for 22 cycles to use as loading control. Other sequences were amplified by PCR for 35–40 cycles in a 20 μl reaction mixture containing cDNA, 10 pmol of each primer, 10 mM dNTP and 0.5 U of Top DNA polymerase. The PCR primers and reagents were purchased from Bioneer. The PCR products were electrophoresed on 1% agarose gels containing ethidium bromide. Quantitative real time PCR was performed using an ABI StepOnePlusTM real time PCR thermal cycler with Power SYBR Green PCR Master Mix according to the manufacturer’s protocol (Life Technology, Carlsbad, CA, USA). The target mRNA levels were normalized to cyclophilin. The experiments were performed in triplicate.
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