Cells were harvested for RNA isolation, 1 mL Trizol reagent (15596026, Invitrogen) was added, followed by the addition of 500 µL chloroform for phase separation and centrifugation at 12000 rpm. The aqueous layer was collected and incubated with 1 mL absolute ethanol for 2 h at -80 °C. The suspension was centrifuged, and the pellet was rinsed with 70% ethanol. Finally, the pellet was reconstituted in 15 µL nuclease-free water, quantified by nanodrop spectrophotometer, and stored at -20 °C. cDNA was synthesized using 1 μg RNA by RevertAid first-strand cDNA synthesis kit (K1622, Thermo Scientific, United States). All the primers were designed by http://www.ncbi.nlm.nih.gov/tool/primerblast and synthesized commercially. quantitative real-time polymerase chain reaction (qPCR) amplification was performed in a triplicate manner using qPCR master mix (A600A, Promega, Madison, WI, United States) for the genes mentioned in Table 1 .
Qpcr master mix
Manufactured by Promega
Sourced in United States
The QPCR Master Mix is a ready-to-use solution for quantitative polymerase chain reaction (qPCR) analysis. It contains all the necessary components, including a DNA polymerase, buffer, and fluorescent dye, to perform qPCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Goscript reverse transcription system, by Promega (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Revertra ace, by Toyobo (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Reverse transcription kit, by Promega (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Mouse anti β actin monoclonal antibody, by Abcam (2 mentions)
M mlv reverse transcription system, by Promega (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Reverse transcription system, by Promega (2 mentions)
High pure rna isolation kit, by Roche (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Goldenstar rt6 cdna synthesis kit, by Tsingke (1 mentions)
Cfx opus real time pcr system, by Bio-Rad (1 mentions)
Oligo dt 12 18 primer, by Thermo Fisher Scientific (1 mentions)
32 protocols using qpcr master mix
1
RNA Isolation and qPCR Analysis
2
RNA Extraction and Quantification Protocol
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RNA extraction was performed with the High Pure RNA Isolation Kit (Roche Applied Science, United States) according to the supplier’s recommendations. The extracted RNAs were quantified at 260 nm using the BioSpec-Nano spectrophotometer (Shimadzu). The cDNAs were synthesized from the total extracted RNAs using random primers and the GoScript Reverse Transcription System (Promega) according to the supplier’s recommendations. For each sample, the amount of RNAs, added to the reaction mixture, was adjusted so that the amount of RNAs was identical for each sample. Thus, despite the difference in sampled volumes and cell density, the same quantity of RNAs from the five samples was used for the reverse transcription. The quantity of the various target cDNAs was quantified by qPCR (qPCR Master Mix, Promega) according to the supplier’s recommendations. Experiences were made using the CFX96 Bio-Rad and the associated software. Various plasmids were constructed by cloning the target genes in the pGEM-T-easy vector (Promega) according to the supplier’s recommendations (Supplementary Table S1 ). They were used for constituting the standard curves for qPCR allowing the quantification of the synthetized cDNAs. Each extracted RNA were analyzed through six technical replicates (three independent reverse transcriptions and two qPCR analyses per cDNAs).
3
Quantitative gene expression analysis
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The total RNA from organoids was extracted by RNeasy Mini Kit (Qiagen). The cDNA was obtained by Revertra Ace (Toyobo). Then, real-time PCR reactions were performed using qPCR Master Mix (Promega) in triplicates on a LightCycler 480 (Roche). The primers of selected gene were shown in Supplementary Table S10 . The experiments were performed with three biological replicates.
4
Quantitative Real-Time PCR for Gene Expression Analysis
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Total RNA was isolated from tissue samples with Trizol (Invitrogen) according to the manufacturer's instructions. RNA was reverse transcribed by M-MLV Reverse Transcriptase (Promega, WI, USA) according to the manufacturer's protocol. Sequence-specific primers for FXR and SHP were, respectively, shown in Table 1 .
Real-time PCR was performed with qPCR Master Mix (Promega, WI, USA) on a Real-Time PCR detection instrument using the SyBr Green detection protocol as outlined by the manufacturer. Briefly, the amplification mixture consisted of 0.5 µM primers, 10 µl of qPCR Master Mix, and 1.5 µl template DNA in a total volume of 20 µl. Samples were amplified with the following program: initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation for 15 s at 95°C and annealing/elongation for 60 s at 60°C. All PCRs were run in triplicate, and control reactions without template were included.
Real-time PCR was performed with qPCR Master Mix (Promega, WI, USA) on a Real-Time PCR detection instrument using the SyBr Green detection protocol as outlined by the manufacturer. Briefly, the amplification mixture consisted of 0.5 µM primers, 10 µl of qPCR Master Mix, and 1.5 µl template DNA in a total volume of 20 µl. Samples were amplified with the following program: initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation for 15 s at 95°C and annealing/elongation for 60 s at 60°C. All PCRs were run in triplicate, and control reactions without template were included.
5
RNA Extraction and qPCR Analysis
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Total RNA was extracted using Trizol reagent (Invitrogen Corp.) and reverse transcribed with a reverse transcription kit (Promega) according to the manufacturer’s protocols. Quantitative PCR was carried out using qPCR Master Mix (Promega). The primer sequences used in this study are listed below. Relative mRNA levels were calculated using the 2−ΔΔCq method and GAPDH was used as an internal control, while U6 was used for miR-142-5p. Primers for Real-Time PCR in this study are listed in Table 1 .
6
Gene Expression Analysis by qRT-PCR
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Total RNA was isolated with TRIzol (Invitrogen, Waltham, MA, USA) and used for cDNA reverse transcription with the Goldenstar RT6 cDNA synthesis kit (Tsingke, Beijing, China). Quantitative PCR analysis of gene transcripts was performed by the qPCR method using qPCR Master Mix (Promega, Madison, WI, USA) and Jena qTOWER3 system with the expression of GAPDH as the endogenous control.
7
qRT-PCR for C. albicans gene expression
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Isolated RNA (1μg) was treated with DNase I (Baseline-ZERO DNase, Lucigen) according to the manufacturer’s instructions and then transcribed into complementary DNA using 50μM of oligo (dT)12-18 primer (Invitrogen), 150 U of Superscript III Reverse Transcriptase (Invitrogen), and 10 U RNase OUT (Invitrogen). The cDNA was then diluted 1:5 and used for q-RT-PCR with the GoTaq qPCR Master Mix (Promega) in a CFX96 thermocycler (BioRad). The expression levels were normalized against the C. albicans ACT1 gene.
For fungal gDNA samples, EFB1 was amplified from 1μl of template with qPCR Master Mix (Promega) in a CFX Opus Real-Time PCR System (BioRad). All primers used are listed inTable 2 . The relative gDNA for each sample was calculated relative to the untreated WT(BWP17) for the respective biological replicate.
For fungal gDNA samples, EFB1 was amplified from 1μl of template with qPCR Master Mix (Promega) in a CFX Opus Real-Time PCR System (BioRad). All primers used are listed in
8
Lipid-induced cellular stress assessment
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Sodium palmitate (Palm), bovine serum albumin (BSA), rapamycin (Rap), bafilomycin A1 (Baf), and carbon tetrachloride (CCl4) were obtained from Sigma‐Aldrich (Buchs, Switzerland). BODIPY 493/503, ASK1 inhibitor (MSC 2032964A), was purchased from Bio‐Techne AG (Zug, Switzerland); Hoechst and Lipofectamine® 2000 were purchased from Invitrogen (Reinach, Switzerland). RNA extraction kits were obtained from Promega (Dübendorf, Switzerland). QPCR master mix was purchased from Promega. SYBR green was obtained from Roche Diagnostics (Rotkreuz, Switzerland).
9
Quantitative Analysis of TGFβ Signaling
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Based on available literature, target prediction results, and gene function, we have chosen the genes encoding isoforms of transforming growth factor β (TGFβ1, TGFβ2, and TGFβ3) and their receptors (TGFβR1, TGFβR2, and TGFβR3) for the gene expression analysis. Reverse transcription was done with the use of GoScript™ Reverse Transcription System (Promega), and resulting cDNA was analysed in quantitative real-time PCR using dye-based qPCR Master Mix (Promega) and set of specific primers spanning exon-exon junction (sequences are presented in Table 1 ). Each Ct value of target mRNA was normalized against an endogenous control, PPIA, which had shown to have the most stable expression in all samples tested (out of 10 potential endogenous controls).
10
Transcriptional Profiling of hMSC Transplants
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At day 14, RNA was isolated from the IVDs tissue transplanted with transfected and non-transfected hMSCs for transcriptional evaluation. Primers for Sox9, TGFβ1, collagen type ll, aggrecan, BMP2, Six1, ADRB2, CXCL2, YKL40, Substance P, MMP-13, COMP2, SOD1, PRDX1, GPX1, and beta-actin (β-actin) were used. The expression of the chondro-specific primers was normalized to β-actin expression. RNA from tissue samples was isolated by the Trizol isolation system (Invitrogen) and purity was determined with spectrophotomer. cDNA was prepared by RevertAidTM First Strand cDNA synthesis kit (K1622, ThermoScientific, USA) followed by qPCR amplifications using qPCR master mix (A600A, Promega, USA).
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