RTL-P assay was performed to detect the 2′-O-methylation level (Nm) of PARP1 according to published methods [13 (link), 14 (link)]. Briefly, 5 μg total RNA was incubated with low (1 μM) or high (1 mM) concentration of dNTPs (Takara) and 1 μl specific RT primers at 65 °C for 5 min and then placed on ice. The reaction was performed using 4 μl M-MLV RT 5 × buffer (PROMEGA), 1 μl 200 U/μl M-MLV Reverse Transcriptase (PROMEGA), 1 μl 0.1 M DTT (PROMEGA), and 1 μl 40 U/μl RNase inhibitor (PROMEGA) at 50 °C for 1 h, and then at 70 °C for 15 min. QRT-PCR was performed on the SYBR Prime X Ex-TAQ Patent II Suite (Takara) using PARP1-specific primers. The relative expression of the gene was calculated by comparing the period threshold (Ct) of the target gene in the experimental group and the control group at low concentrations according to the 2−ΔCt method.
M mlv rt 5 buffer
Manufactured by Promega
Sourced in United States
M-MLV RT 5× Buffer is a concentrated buffer solution designed for use with Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) in reverse transcription reactions. The buffer provides the necessary ionic conditions and cofactors to facilitate the enzymatic conversion of RNA to complementary DNA (cDNA).
Automatically generated - may contain errors
Lab products found in correlation
M mlv reverse transcriptase, by Promega (7 mentions)
Dntps, by Takara Bio (2 mentions)
Sybr prime x ex taq patent 2 suite, by Takara Bio (2 mentions)
0.1 m dtt, by Promega (2 mentions)
Rnase inhibitor, by Promega (2 mentions)
Dntp mix, by Promega (2 mentions)
M mlv rt, by Promega (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Dntp mixture, by Promega (1 mentions)
Oligo dt 15 primer, by Promega (1 mentions)
Thunderbird sybr qpcr mix, by Toyobo (1 mentions)
Cfx96 optics module, by Bio-Rad (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Random primer, by Promega (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Roche (1 mentions)
9 protocols using m mlv rt 5 buffer
1
Quantitative Analysis of RNA 2'-O-Methylation
2
Reverse Transcription and qPCR Protocol
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Reverse transcription was performed using 1.2 μl M-MLV-RT and 8 μl M-MLV-RT 5× buffer (Promega), 4 μl 5 mM dNTPs, 4 μl Random Primers at 100 ng/μl, 2 μl DTT 0.1 M, and 1 μg RNA in a final volume of 40 μl. The reaction was run in the C1000 Touch Thermal Cycler from Bio-Rad. The samples were incubated at 37°C for 60 min, then at 95°C for 60 s, and next immediately placed at 4°C.
Quantitative polymerase chain reaction was performed in the CFX96 Real-Time system from Bio-Rad. For the reaction, 10 μl IQ Syber Green mix from Bio-Rad, 0.4 μl of each primer at 15 μM, and 2 μl of the DNA sample at 0.01 μg/μl were mixed in a final volume of 20 μl. The mixture was first incubated at 95°C for 3 min, and then at 95°C for 15 s, 60°C for 15 s, and 72°C for 25 s for 34 cycles. The PCR ended after 1 min at 95°C and 1 min at 65°C. The results were analyzed with Bio-Rad CFX-manager software. GAPDH levels were evaluated in all cases as a reference. Only the samples with similar GAPDH amplification were analyzed further. The primers used are listed in Table S3 in Supplementary Material and were designed using the Primer3 program2 . Initial setups included GC percentage between 30 and 70%, product size from 150 to 300 bp and primer length between 18 and 27 nt.
Quantitative polymerase chain reaction was performed in the CFX96 Real-Time system from Bio-Rad. For the reaction, 10 μl IQ Syber Green mix from Bio-Rad, 0.4 μl of each primer at 15 μM, and 2 μl of the DNA sample at 0.01 μg/μl were mixed in a final volume of 20 μl. The mixture was first incubated at 95°C for 3 min, and then at 95°C for 15 s, 60°C for 15 s, and 72°C for 25 s for 34 cycles. The PCR ended after 1 min at 95°C and 1 min at 65°C. The results were analyzed with Bio-Rad CFX-manager software. GAPDH levels were evaluated in all cases as a reference. Only the samples with similar GAPDH amplification were analyzed further. The primers used are listed in Table S3 in Supplementary Material and were designed using the Primer3 program
3
Quantitative Analysis of Inflammatory Markers
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Total RNA was isolated from each group using the QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using RNase Inhibitor, M-MLV RT 5× Buffer, M-MLV Reverse Transcriptase, Oligo(dT)15 primer, and dNTP mixture manufactured by Promega (Madison, WI, USA), and quantitative real-time PCR was performed using the THUNDERBIRDTM SYBR qPCR Mix (Toyobo, Osaka, Japan) on the CFX96TMOptics Module (Bio-Rad, Hercules, CA, USA), with the following primers: HRV5′-NCR-up: 5′-TCC TCC GGC CCC TGA ATG-3′, HRV5′-NCR-down: 5′-GAA ACA CGG ACA CCC AAA G-3′, CCL2-up: 5′-TTA AAA ACC TGG ATC GGA ACC AA-3′, CCL2-down: 5′-GCA TTA GCT TCA GAT TTA CGG GT-3′, IL-6 up: 5′-CTG GAG TCA CAG AAG GAG TGG-3′, IL-6 down: 5′-GGT TTG CCG AGT AGA TCT CAA-3′, CXCL1 up: 5′-TGA GCT GCG CTG TCA GTG CC-3′, CXCL1 down: 5′-GCG TTC ACC AGA CGG TGC CA-3′, TNF-α up: 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′, and TNF-α down: 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′, IL-1β up: 5′-GTT GAC GGA CCC CAA-3′, IL-1β down: 5′-AAG ATA AGG TCC ACG GGA AAG ACA C-3′, β-actin up: 5′-CCA TCA TGA AGT GTG ACG TGG-3′, and β-actin down: 5′-GTC GCC TAG AAG CAT TTG CG-3.
4
RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis
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Total RNA was extracted with TRIzol (15596026, Invitrogen, USA) reagent following the manufacturer's instructions and quantified the RNA using Nanodrop 2000 (Thermo Fisher Scientific, MA, USA). For lncRNA and mRNA quantifying, cDNA was synthesized using the M-MLV Reverse Transcriptase (m1705, Promega, USA) and dNTP Mix (u1511, Promega, USA) and M-MLV RT 5× Buffer (M531A, Promega, USA) and Random Primers (C1181, Promega). And then FastStart Universal SYBR Green Master (ROX) (04913914001, Germany) was used for qRT-PCR. The detection of miRNAs was used the miDETECT A Track miRNA qRT-PCR Starter Kit (C10712-1, RIBBIO). RPS18 and U6 were employed as endogenous controls for lncRNA/mRNA qRT-PCR and miRNA qRT-PCR, respectively. Primer sequences are listed in Table 1 . The primer sequences of U6 were not available from the provider. The relative expression of lncRNAs, mRNAs, and miRNAs was calculated by the 2−△△Ct method. The qPCRs were all repeated three times for the three matched samples.
5
Quantifying Bacterial Gene Expression
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The abundance of bacterial 16S rRNA genes was quantified using qPCR, with primers Eub338F and Eub518R (Supplementary Table 7 ). Primers used for quantifying genes involved in DMSP synthesis and degradation are listed in Supplementary Table 7 . The construction of qPCR standards and the qPCR assay was performed as described in a previous study46 (link). For reverse-transcription qPCR, 9 µl of purified RNA was mixed with 1 µl random hexamer primers (Invitrogen). The reaction system was incubated at 70 °C for 5 min and cooled on ice, before mixing with 1 µl dNTPs (10 mM), 4 µl of M-MLV RT 5× buffer (Promega), 0.8 µl of M-MLV reverse transcriptase (Promega), 0.4 µl of RNase inhibitor (40 U µl−1, Roche), and 3.8 µl of Diethyl Pyrocarbonate (DEPC) water. This was then incubated at 42 °C for 1 h and the obtained cDNA was stored at −80 °C.
6
SNORD89 2'-O-methylation Detection
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To detect the 2′-O-methylation (Nm) of SNORD89, RT-PCR was performed [32 (link), 37 (link)]. The total reaction system was 20 μl. Containing 5 μg total RNA, a low (1 μM) or high (1 mM) concentration dNTPs (TaKaRa) and 1 μl specific RT primers (PF) were denatured at 65 °C for 5 min and then placed on ice. According to the instruction, 4 μl M-MLV RT 5×buffer (PROMEGA), 1 μl 200 u/μl M-MLV Reverse Transcriptase (PROMEGA), 1 μl 0.1 M DTT (PROMEGA), and 1 μl 40 u/μl RNase inhibitor (PROMEGA) were mixed at 50 °C for 1 h, and then heated at 70 °C for 15 min. cDNA is used as a template for QRT-PCR using the SYBR Prime X Ex-TAQ Patent II Suite (Takara). Finally, the relative expression of the gene was calculated by comparing the period threshold (Ct) of the target gene in the experimental group and the control group at low concentrations according to the 2−ΔCt method.
7
Quantitative Analysis of Differentiation Markers
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The cell pellet was obtained after being differentiated for 14 days. Total RNA from hASC were purified by Rneasy Mini Kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. cDNA was synthesized from 1 μg of total RNA by reverse transcription using dNTP mix, Rnase inhibitor (Enzynomics Co., Ltd., Daejeon, Korea), M-MLV Reverse Transcriptase, M-MLV RT 5× Buffer (Promega Corporation, Madison, WI, USA) and random oligo primers. qRT-PCR was performed using SYBR Green 2× Mastermix kit (Messenger of biotechnology, Hanam, Korea) on a BioRad CFX96 Real-Time PCR Detection System instrument (Bio-Rad Laboratories, Hercules, CA, USA) under the following conditions: 10 min at 95 °C, and then 40 cycles of 15 s at 95 °C for denaturation and 60 s at 60 °C for annealing and elongation. Specificity of products was verified by melting curve analysis. The delta-delta-Ct method was employed to determine the relative gene expression level of gene of interest normalized to the house-keeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers used for differentiation markers and Nrf2 pathway genes are listed in Table 1 .
8
cDNA Synthesis from RNA Templates
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First strand synthesis was performed in 25 μL reaction volumes using 5 μL of MMLV-RT 5× buffer (Promega, Madison, WI, USA), 1.25 μL of dNTPs (10 mM) (Promega), 0.625 μL of recombinant RNasin (Promega), 1 μL of random hexamer primers (Promega) and 1 μg of RNA in 16.125 μL. Prior to the addition of 1 μL of MMLV-RT enzyme (Promega), the reaction was incubated at 65 °C in order to denature double stranded RNA. Following the addition of the MMLV-RT, the reaction was incubated at 37 °C for 1 h. Second strand synthesis was performed immediately after first strand synthesis. Single-stranded cDNA was incubated at 94 °C for 2 min, followed by 10 °C for 5 min. Following incubation, 2 μL Sequenase buffer (Thermo Fisher, Waltham, MA, USA), 0.3 μL Sequenase (Thermo Fisher) and 7.7 μL molecular grade water (Thermo Fisher) were added. Following this, reactions were incubated at 37 °C for 8 min, 94 °C for 2 min and 10 °C for 5 min. During incubation at 10 °C, 1.2 μL 1:4 (Sequenase:Sequenase dilution buffer (Thermo Fisher)) diluted Sequenase was added to reactions and incubated at 37 °C for 8 min and 94 °C for 8 min prior to transfer to ice.
9
mRNA Expression Analysis by qRT-PCR
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For mRNA analysis cDNA conversion was carried using 2 μg of RNA, 2 μl of Oligo (dT) 15 primer (Cat. No. C1101, Promega), 1 μl of M-MLV reverse transcriptase (Cat. No. M1701, Promega), 5 μl of M-MLV RT 5× buffer (Cat. No. M531A, Promega), 0.2 μl of RNasin (Cat. No. N2515, Promega), 0.5 μl of dNTP mix (Cat. No. U1240, Promega) and nuclease-free water in a 25 μl reaction volume. PCR analysis was carried out using 1 μl of 1:5 diluted cDNA in a reaction mixture containing 5 μl of Fast SYBR green 2× Master mix (Cat No. 4385612, Applied Biosystems, Life technologies), 0.5 μl each of 10 μm forward and reverse primer and the total volume adjusted to 10 μl using RNase-free water. The reaction was carried out in Applied Biosystems 7900HT Fast Real-Time PCR machine. The primer sequences are listed in Table 2 .
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