Total RNA was isolated with Trizol Reagent from the untreated MG-63 cells and MG-63 cells treated with BDCe fraction (37.53 µM), as per the manufacturer’s protocol. The RNA samples were dissolved with TE buffer and incubated at 60 °C for 5 min. DNA impurities were removed with DNase-I solution, and the resulting solution was incubated for 30 min at 37 °C. Finally, the quantification of RNA was performed using the Nano-Drop spectrophotometer (Thermo, Waltham, MA, USA). Further, equal concentrations of RNAs were used for cDNA preparation using the iScriptTM cDNA (Biorad) synthesis kit as per the manufacturer’s protocol. cDNA was used to perform RT-qPCR in a one-step RT-qPCR system using iQ SYBR Supermix (Biorad). The genes used as the biomarkers for RT-qPCR analysis and their primer sequences (Supplementary Table S4 ) were the following: p53, Bcl-2, CDK2, Cyclin E, and Caspase3. The expression of each gene was quantified by using threshold cycle method 2−ΔΔCt ± SEM.
Iq sybr supermix
Manufactured by Bio-Rad
Sourced in United States, France
IQ SYBR Supermix is a ready-to-use solution for real-time PCR amplification and detection using SYBR Green fluorescence. It contains all the necessary components, including DNA polymerase, dNTPs, buffer, and SYBR Green I dye.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (15 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (8 mentions)
Iq5 real time pcr detection system, by Bio-Rad (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Ampure xp pcr purification system, by Beckman Coulter (4 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (4 mentions)
High capacity cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Rneasy plus mini kit, by Qiagen (3 mentions)
Icycler real time pcr machine, by Bio-Rad (3 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Cfx96 real time system device, by Bio-Rad (3 mentions)
Cfx manager software, by Bio-Rad (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Miseq 300, by Illumina (2 mentions)
Cfx connect qpcr system, by Bio-Rad (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Cfx96 real time pcr machine, by Bio-Rad (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
66 protocols using iq sybr supermix
1
Quantifying Apoptosis-Related Genes in MG-63 Cells
2
Validating Gfi1 and Lsd1 Gene Expression
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To validate expression of Gfi1 and Lsd1 target genes, mRNA was isolated from cells using an RNeasy Plus Mini kit (Qiagen). RNA was reverse transcribed to cDNA using iScript Reverse Transcription Supermix (Bio-Rad, cat #1708841), and duplicate no RT reactions were also prepared to confirm absence of genomic contamination. Then qPCR reactions were performed in triplicate using iQ SYBR Supermix (Bio-Rad, cat #1708882) on the Bio-Rad C1000 Thermocycler and CFX96/CFX384 systems. Relative gene expression was calculated using the ∆∆CT method and normalized to Actin. 95% confidence intervals for each sample were calculated using the sum of squares method.
3
16S rRNA Gene Amplicon Sequencing from Mouse Stool
4
Quantifying Rat Brain mRNA Levels
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Quantification of mRNA was conducted using reverse transcription PCR and primers specifically designed for rat mRNAs Lsd1, Lsd1 + 8a, and Bdnf exon IV (Table 1 ). RNA was isolated from amygdala tissue as described previously (Kyzar et al., 2017 (link), 2019 (link)) and then reverse transcribed in duplicate using mixed random primers and MuLV reverse transcriptase (Life Technologies). Quantitative real-time PCR was performed using either the Mx3000P qPCR system (Agilent Technologies) and SYBR Green master mix (Fermentas) or a CFX Connect qPCR system with iQ SYBR SuperMix (Bio-Rad). Data were analyzed using the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)). Hprt1 was used as a reference gene (Kyzar et al., 2017 (link), 2019 (link)). Data are represented as fold change compared to control AIS groups.
5
Quantifying Defense-Related Gene Expression
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The transcript levels of defense-related genes PI-I, PI-II, Mi-1.2, PR-1a, and PR-1b in samples were quantified by real-time quantitative RT-PCR (qRT-PCR). The qRT-PCR was carried out on an ABI 7500 Real Time PCR System with a 96-well rotor. The amplification reactions were performed in a final volume of 20 μL that contained 10 μL of iQ SYBR supermix (Bio-Rad), 0.8 μL of forward primer (5 µM) and reverse primer (5 µM) pairs (SI Appendix, Table S1 ), and 2 μL of cDNA first-strand template. Thermal cycling conditions were 5 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 15 s at 55–62 °C, and 30 s at 72 °C. Subsequently, a melting curve was recorded between 60 °C and 95 °C with the hold every 5 s. Primers used for qRT-PCR are listed in SI Appendix, Table S1 . All reactions were run in duplicate technical replicates, and average values were used in the analysis. Normalized gene expression was calculated using the 2−∆Ct method with GAPDH as an endogenous control gene, and values were subsequently log2 transformed for data analysis.
6
RNA Isolation and Quantitative PCR
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RNA was isolated from frozen liver and cecum tissue or fresh BMDM cells using an E.Z.N.A. Total RNA Kit I (Omega Bio-tek, Norcross, GA), per instructions, except for TRIzol reagent (ThermoFisher) replaced TRK Lysis Buffer. RNA quality and quantity were determined by the Experion Automated Electrophoresis System (Bio-Rad). cDNA was synthesized from 1 µg RNA using iScript reaction mix and reverse transcriptase (Bio-Rad). The cDNA synthesis reaction was incubated at 25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min. Quantitative PCR was performed with primer sets (Supplementary Table 2 ) for genes of interest and reference genes (designed with NCBI’s Primer-BLAST) and iQ SYBR Supermix (Bio-Rad) using the following conditions: initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Reactions were run in duplicate on an iQ5 Real Time PCR detection system (Bio-Rad) along with a no-template control per gene. Validation experiments were performed to demonstrate that efficiencies of target and reference genes were approximately equal. Data were normalized to 18S rRNA and Gapdh using the comparative Ct method.
7
Optimized Protein and RNA Extraction Workflow
8
Bacterial RNA Isolation and Quantification
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RNA isolation, cDNA synthesis and RT-PCR were performed as described previously (Korir et al., 2014 (link); Korir, 2016 ). Briefly, RNA samples were collected from bacterial culture in liquid medium by adding two volumes of RNAprotect Bacteria Reagent (Qiagen) or from bacteria inside macrophages by washing the cells twice with PBS then adding 1 ml RNAprotect Bacteria Reagent directly to the cells. RNA was then extracted using the RNeasy minikit (Qiagen) using the “Enzymatic Lysis, Proteinase K Digestion, and Mechanical Disruption of Bacteria” protocol in the RNAprotect handbook. Residual genomic DNA was removed using the Turbo DNA-free kit (Ambion). The iScript Select cDNA synthesis kit was used to synthesize cDNA using random primers. As a control to test for DNA contamination, samples were processed without reverse transcriptase. RT-PCR analysis was performed using the iQ SYBR Supermix (Bio-Rad) and gene specific primers (Table 2 ). Relative fold change in gene expression was calculated using the 2-ΔΔCt method and relative transcript level was calculated using 2-ΔCt method; gyrA was used as the internal control for both (Schmittgen and Livak, 2008 (link)).
9
Myogenic Differentiation Markers Analysis
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Myosin heavy chain (MyHC) mRNA content was measured from day 0 to day 5 of myogenic induction, as were nanog and Oct4, identified markers of mesenchymal stem cell pluripotency (23 (link)). Gene names and primer sequences are listed in Supplementary Table 3 . Cells were rinsed twice with PBS, then harvested in Buffer RLT (Qiagen, Valencia, CA). Total RNA was isolated using an RNeasy Plus Mini Kit (Qiagen). cDNA was transcribed from 200 ng total RNA using an iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed using primer sets for genes of interest, RPL13a and ubiquitin C as reference genes, and iQ SYBR Supermix (Bio-Rad, Hercules, CA), as described elsewhere (24 (link)). Reactions were run in duplicate on an iQ5 Real-Time PCR Detection System (Bio-Rad), along with a no-template control per gene. RNA expression data were normalized to reference genes using the comparative threshold cycle method.
10
Quantifying Gene Expression in Muscle Atrophy
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