The total RNA from was extracted from the anterior tibial muscle or from serum of burn and control patients with TRIZOL reagents (15596-026, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and quantified with ultraviolet spectrophotometry (DU-800, Beckman, Fullerton, CA, USA). The expression levels of indicated miRNAs were done using customized Megaplex™ Primer Pools and TaqMan miRNA arrays (ThermoFisher Scientific). Data was normalized to U6 snRNA expression and analyzed by the -ΔΔCt method.
Megaplex primer pools
Manufactured by Thermo Fisher Scientific
Sourced in United States
Megaplex Primer Pools are a collection of pre-designed and optimized primer sequences for use in multiplexed PCR (polymerase chain reaction) applications. These primer pools are designed to amplify multiple target sequences simultaneously, enabling efficient and high-throughput analysis of samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Megaplex preamp primers, by Thermo Fisher Scientific (2 mentions)
Taqman array human microrna a card, by Thermo Fisher Scientific (2 mentions)
Taqman mirna arrays, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Du800, by Beckman Coulter (1 mentions)
Genesifter software, by PerkinElmer (1 mentions)
7900ht thermocycler, by Thermo Fisher Scientific (1 mentions)
Dataassist, by Thermo Fisher Scientific (1 mentions)
Sds 2, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (1 mentions)
Expression suite software, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
7 protocols using megaplex primer pools
1
Burn-Induced Muscle miRNA Expression
2
miRNA Expression Profiling in PBMCs
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The miRNA expression profiles were determined with a TaqMan Array Human microRNA Card A (Thermo Fisher Scientific, Waltham, MA, USA). Ninety nanograms of total RNA was used as an input in each RT reaction. The RT reaction and pre-amplification step was set up according to the manufacturer’s recommendations. miRNAs were reverse transcribed with the Megaplex Primer Pools (Human Pools A version 2.1; Thermo Fisher Scientific). RT reaction products from the PBMC samples were further amplified with Megaplex PreAmp primers (Primers A version 2.1; Thermo Fisher Scientific). Quantitative RT-PCR was performed on an Applied Biosystems 7900HT thermocycler according to the manufacturer’s recommended program. With the use of SDS 2.4 software and DataAssist (Thermo Fisher Scientific), the expression of miRNA was calculated based on cycle threshold (Ct) values normalized by those of RNU6B. Data analysis was done using GeneSifter® software (Perkin Elmer, Waltham, MA, USA). P-values of less than 0.05 were considered to indicate a statistically significant difference, and the Benjamini–Hochberg algorithm was used for estimation of false discovery rates, as we have reported previously.19 (link)
3
Profiling Liver microRNA Expression
4
RNA Isolation from Human Muscle
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Human skeletal muscle (∼10 mg) was freeze-dried before microscopical removal of fat, blood, and connective tissue. A Trizol method was used for RNA extraction according to the manufacturer’s recommendations (Life Technologies, Stockholm, Sweden). Total RNA concentration was quantified spectrophotometrically at an absorbance of 260 nm (NanoDrop ND-1000 Spectrophotometer; Thermo Fisher Scientific, Waltham, MA). The integrity and purity of the RNA was verified by measuring spectrophotometrically at A260/A280 (>1.8). For quantification of target gene expression, RNA (1 μg) was reverse transcribed to cDNA using the High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA). To quantify microRNA expression, RNA (350 ng) was reverse transcribed using Megaplex Primer Pools (Human Pools) Kit or custom Primer Pool (Applied Biosystems). In both analyses, a reaction without reverse transcriptase was included as a control. The cDNA template was stored at −20°C until subsequent analysis.
5
MicroRNA Profiling via Taqman OpenArray
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MiRNA profiling was conducted using the Taqman OpenArray microRNA platform (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions with minor adjustments. Then, 10 ng of RNA was retrotranscribed using Megaplex Primer Pools (Applied Biosystems), and cDNA was pre-amplified using Megaplex PreAmp Primer pools (Applied Biosystems) as previously described [14 (link)]. The miRNA profiling was conducted on QuantStudio 12 k flex Real-Time PCR system (Applied Biosystems). ExpressionSuite Software (Applied Biosystems) was used to analyze the data by applying the relative quantification method. Low expressed miRNAs, with Crt (relative threshold cycle) higher than 27 or low amplification score, were excluded from the analysis. The expression of each miRNA was normalized globally to the mean expression of all the expressed miRNAs. The relative fold change (FC) of each miRNA was normalized to the mean of the control group (healthy controls).
6
Age-dependent miRNA profiling in Down Syndrome
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MiR microfluidic card, TaqMan Array Human MicroRNA A Card (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA), for the measurements of 377 human miRs, was applied to identify miRprofiles in discovery phase testing 6 plasma samples from 3 young DS females (age: 30, 30 and 34 years old) and 3 elderly DS females (age: 63, 66 and 68 years old).
RNA was converted to cDNA by priming with a mixture of looped primers and then pre-amplified using the MegaPlex primer pools (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer's instructions. The profiling was run on the Applied Biosystems 7900 HT real-time PCR instrument.
MiRs profiling was normalized with the median of the overall miR expression on each array (ΔCt). Only miRs expressed in at least five of the six analysed samples were selected and Ct values < 32 were established as cut-off. Fold-change (2 -ΔΔCT ) was calculated based on the estimated mean difference and fold changes ≥ 2 and ≤ -2 were selected.
RNA was converted to cDNA by priming with a mixture of looped primers and then pre-amplified using the MegaPlex primer pools (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer's instructions. The profiling was run on the Applied Biosystems 7900 HT real-time PCR instrument.
MiRs profiling was normalized with the median of the overall miR expression on each array (ΔCt). Only miRs expressed in at least five of the six analysed samples were selected and Ct values < 32 were established as cut-off. Fold-change (2 -ΔΔCT ) was calculated based on the estimated mean difference and fold changes ≥ 2 and ≤ -2 were selected.
7
miRNA Profiling in Liver Transplant
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Human miR microfluidic card, TaqMan Array Human MicroRNA A Card (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA), enabling the quantitation of 377 human miRs, was used to identify miR-profiles in 6 serum samples from 3 recipients before and after LT (the first three patients in Table 1 are highlighted in bold).
RNA was converted to cDNA by priming with a mixture of looped primers and then pre-amplified using the MegaPlex primer pools (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The profiling was performed using an Applied Biosystems 7900 HT real-time PCR instrument.
The MiR profiling was normalized using the median of the overall miR expression on each array (ΔCt). Only the miRs expressed in all of the samples were selected for analyses, and Ct values ≤ 30 were established as the cut-off. The fold change (2−ΔΔCT) was calculated based on the estimated mean difference between vascular groups. Fold changes ≥ 2 and ≤−2 were selected.
RNA was converted to cDNA by priming with a mixture of looped primers and then pre-amplified using the MegaPlex primer pools (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The profiling was performed using an Applied Biosystems 7900 HT real-time PCR instrument.
The MiR profiling was normalized using the median of the overall miR expression on each array (ΔCt). Only the miRs expressed in all of the samples were selected for analyses, and Ct values ≤ 30 were established as the cut-off. The fold change (2−ΔΔCT) was calculated based on the estimated mean difference between vascular groups. Fold changes ≥ 2 and ≤−2 were selected.
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