Cells were plated the day before the experiments onto 6-well plates (3x105) or 12-well plates (1x105). Total RNA was isolated using RNeasy Kit (Qiagen). miRCURY™ RNA Isolation Kit (Exiqon, Denmark) was used for microRNAs extraction. RNA isolation was carried following manufacturer’s protocols. RNA was quantified using the fluorimeter Qubit 2.0 (Life Technologies) following manufacturer’s instructions or Nanodrop (Thermo). Reverse transcription of RNA was performed using Quantitect-Reverse transcription kit (Qiagen) or miScript PCR kit (Qiagen) using 300-500 ng of total RNA. Real time qPCR was performed using Quantitect Syber Green master mix (Qiagen) or Taqman universal mix (Life Technology) on a Step One Plus real-time PCR system (Life Technology). Experiments were analysed using the software Expression Suite (Life Technology) and StepOne software 2.3 and Relative quantification (RQ) with max and min values (RQ max and RQ min) were calculated using S.D. algorithm. Statistical analysis was performed using Expression Suite software on at least three independent cultures. Housekeeping genes used for internal normalisation are β-Actin for mRNA and Snord95 Snord61 and RNU6B, for miRNAs. The primers were designed using ProbeFinder- Roche or purchased by Qiagen and are listed in SI Table 1 .
Taqman universal mix
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
TaqMan Universal Master Mix is a ready-to-use, real-time PCR mix that contains all the necessary components for quantitative gene expression analysis, including a proprietary hot-start DNA polymerase, dNTPs, MgCl2, and a passive reference dye. The mix is optimized for use with TaqMan Assays and can be used with a variety of sample types and instrumentation platforms.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Mircury rna isolation kit, by Qiagen (2 mentions)
Miscript pcr kit, by Qiagen (2 mentions)
Quantitect syber green master mix, by Qiagen (2 mentions)
Expression suite, by Thermo Fisher Scientific (2 mentions)
Qubit 2, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Dna rna mini kit, by Qiagen (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Trizol reagent protocol, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mxpro qpcr software, by Agilent Technologies (1 mentions)
Mx3005p platform, by Agilent Technologies (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
6 protocols using taqman universal mix
1
Quantitative Gene Expression Analysis
2
Quantitative Gene Expression Analysis
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Cells were plated the day before the experiments onto 6-well plates (3x105) or 12-well plates (1x105). Total RNA was isolated using RNeasy Kit (Qiagen). miRCURY™ RNA Isolation Kit (Exiqon, Denmark) was used for microRNAs extraction. RNA isolation was carried following manufacturer’s protocols. RNA was quantified using the fluorimeter Qubit 2.0 (Life Technologies) following manufacturer’s instructions or Nanodrop (Thermo). Reverse transcription of RNA was performed using Quantitect-Reverse transcription kit (Qiagen) or miScript PCR kit (Qiagen) using 300-500 ng of total RNA. Real time qPCR was performed using Quantitect Syber Green master mix (Qiagen) or Taqman universal mix (Life Technology) on a Step One Plus real-time PCR system (Life Technology). Experiments were analysed using the software Expression Suite (Life Technology) and StepOne software 2.3 and Relative quantification (RQ) with max and min values (RQ max and RQ min) were calculated using S.D. algorithm. Statistical analysis was performed using Expression Suite software on at least three independent cultures. Housekeeping genes used for internal normalisation are β-Actin for mRNA and Snord95 Snord61 and RNU6B, for miRNAs. The primers were designed using ProbeFinder- Roche or purchased by Qiagen and are listed in SI Table 1 .
3
SNP Genotyping of TNPO3 Variants
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Total genomic DNA and RNA were isolated from PBMCs using DNA/RNA Mini Kit (Qiagen). SNP genotyping assay was designed for detecting simultaneously TNPO3_wt and TNPO3_mut forms using the following primers and probes: forward primer (5´-GCGAGACTTCACCAGGTTGTT-3´); reverse primer (5´-CTGGGTGACAGGCACAGT-3´); TNPO3_wt probe: (TNPO3 deletion-V, VIC, 5´-CAGGAGTGTGAGCTATCGA-3´); and TNPO3_mut probe (TNPO3 deletion-M, FAM, 5´-AGGAGTGTGAGCATCGA-3´). cDNA was synthesized from 200 ng of RNA by using GoScript Reverse Transcription System (Promega), following manufacturers’ instructions. RT cycling conditions were as follows: 5 min at 25°C; 1h at 45°C; and 15 min at 70°C. SNP genotyping was also performed using 50 ng of genomic DNA and TaqMan Universal Mix (Applied Biosystems). Analyses were performed in triplicate per sample using StepOne Real-Time PCR system (Applied Byosistems) with standard cycling conditions. Results for the allelic discrimination were represented as ΔRn, being Rn the ratio between the fluorescent emission intensity of the reporter dye and the passive dye.
4
Quantitative RT-PCR Analysis of CRBN Gene
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Quantitative RT-PCR analysis was performed with the Applied Biosystems 7900HT Fast Real-Time PCR system using Taqman detection according manufacturer’s instructions. Briefly, total RNA was isolated from AML cells using the standard TRIzol reagent protocol (Life Technologies). 2 µg of the obtained RNA was reverse transcribed using oligo(dT). Each PCR assay was performed in a 25 µl reaction containing 2× TaqMan universal Mix (Applied Biosystems) reagent, 20× primers and 2 µl cDNA (equivalent to 40 ng of total RNA). Ct (threshold cycle) was calculated for each assay (Sequence Detection System Software 2.4.1, Applied Biosystems). Data are normalized using GAPDH as endogenous control (ΔCt = CtCRBN − CtGAPDH). Results are presented as R = 2−ΔCt. CRBN and GAPDH TaqMan Gene Expression assays were purchased from Applied Biosystems.
5
SERT Gene SNP Genotyping in MMVD
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DNA was isolated from 200 μL EDTA‐anticoagulated blood with a DNA extraction kit (Epicenter Kit, Nordic Biolabs, Sweden) and kept at −20°C. Three selected SNPs in the SERT gene with possible functional impact and association with MMVD were: c.814insG (p.Lys272Arg), c.1193delT (p.Val397Gly), c.1324G>A (p.Gly442Ser; Table 1 ).19 Single nucleotide polymorphism genotyping was performed by TaqMan assays established based on sequence information extracted from Ensembl, comprising 100 to 200 bp sequences on both sides of the polymorphisms. Primers and probes were designed for wild type and mutated sequences, respectively, by Thermo Fischer Scientific, Denmark using VIC for wild type alleles and FAM for variant alleles. TaqMan real‐time PCR was carried out as follows on the Mx3005P platform (Agilent, Glostrup, Denmark): 1 μL of genomic DNA (20‐25 ng/μL) was amplified in a total volume of 10 μL reaction mastermix containing 5.0 μL TaqMan Universal Mix, 0.25 μL TaqMan enzyme (Thermo Fischer Scientific, Denmark) and 3.75 μL H2O. PCR conditions were: 1 cycle of 50°C for 2 minutes, 1 cycle of 95°C for 10 minutes, and 45 cycles of 92°C for 20 seconds followed by 60°C for 1 minute. Water was included as negative control and results were analyzed by MxPro qPCR Software (Agilent, Glostrup, Denmark).
6
Cryopreserved Human Hepatocyte Spheroid Analysis
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Cryopreserved human hepatocytes were obtained from Bioreclamation IVT (BioIVT, Westbury, NY, USA), KaLy‐Cell (Plobsheim, France), and Lonza (Basel, Switzerland), and 3D human hepatocyte spheroids prepared as described.14 The donors used were: SYF, GID, HJK (BioIVT), HUM201221 (Lonza), and S1506T (KaLy Cell). Total RNA was isolated from 48 pooled human hepatic spheroids using the QIAzol Lysis Reagent (Qiagen, USA) and the RNA concentration was measured at a Qubit 4 fluorometer (Thermo Fisher Scientific). Following the RNA extraction, cDNA synthesis was performed using the SuperScript III reverse transcriptase (Thermo Fisher Scientific) and gene expression was analyzed by reverse transcription polymerase chain reaction (RT‐qPCR) using a TaqMan Universal mix and TaqMan probes (obtained from Thermo Fisher; Table S1
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