In our discovery phase we conducted genome-wide miRNA profiling using Taqman MicroRNA Arrays, also known as Taqman Low Density Array (TLDA) ‘Pool A’ Card version 3.0, due to their established use for miRNA expression analysis in FFPE tissue [28 (link),29 (link)]. This 384-microfluidic array was designed to perform quantitative reverse transcriptase (qRT-PCR) reactions simultaneously (Applied Biosystems, Austin, TX, USA) on 378 mature miRNAs (and 6 endogenous controls) that tend to be functionally defined. Using 20 nanograms (ng) of total RNA as input, cDNA was synthesized with highly multiplexed Megaplex RT primers, pre-amplified with Megaplex PreAmp Primers, mixed with TaqMan Universal PCR Master Mix (Applied Biosystems), and loaded onto TLDA cards for expression analysis.
Megaplex rt primers
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Megaplex RT Primers is a set of primers designed for use in reverse transcription (RT) reactions. The primers enable the simultaneous conversion of multiple RNA targets into cDNA, which can then be used in downstream applications such as real-time PCR or other gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (24 mentions)
Megaplex preamp primers, by Thermo Fisher Scientific (19 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (9 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (7 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (5 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (4 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (4 mentions)
Taqman array microrna card, by Thermo Fisher Scientific (3 mentions)
Rq manager 1, by Thermo Fisher Scientific (3 mentions)
Sds 2, by Thermo Fisher Scientific (3 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Taqman array human microrna a card, by Thermo Fisher Scientific (3 mentions)
Thermo fisher cloud, by Thermo Fisher Scientific (3 mentions)
Taqman microrna rt kit, by Thermo Fisher Scientific (3 mentions)
Rq manager software, by Thermo Fisher Scientific (2 mentions)
Mirvana kit, by Thermo Fisher Scientific (2 mentions)
Dataassist v3, by Thermo Fisher Scientific (2 mentions)
Taqman preamp master mix kit, by Thermo Fisher Scientific (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
54 protocols using megaplex rt primers
1
Genome-Wide miRNA Profiling in FFPE Tissue
2
Plasma miRNA Profiling Using TaqMan Assays
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TaqMan® Array Human MicroRNA A and B Cards v2.0 (Cat no 4400238, Applied Biosystems) were employed to examine the expression of 667 miRNAs in 48 plasma samples in the discovery cohort. Reverse transcription and quantitative PCR (qPCR) were performed on equal volumes of RNA from each sample according to the manufacturer’s instructions using TaqMan® MicroRNA Reverse Transcription Kit (Cat no 4366596, Applied Biosystems) and Megaplex RT Primers to convert the miRNAs to cDNA, TaqMan® PreAmp Master Mix (Cat no 4391128, Applied Biosystems) and Megaplex PreAmp Primers for a preamplification step before real-time analysis. qPCR was performed using TaqMan® Universal Master Mix II, no UNG (Cat no 4440048, Applied Biosystems) on the 7900HT Fast Real-Time PCR system (Applied Biosystems). The Sequence Detector System software version 2.2.2 was utilised to generate study files using a fixed threshold value of 0.1 for statistical analysis (accession no: GSE67075).
3
MiRNA Expression Profiling in BM-MSCs
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The conditioned medium from Normal BM-MSCs (control) or the BM-MSCs co-cultured with ovarian cancer (BM-MSCs) was collected for total RNA extraction using TRIzol (Roche Applied Science). A three-step procedure was performed to profile the miRNAs. First, for cDNA synthesis from the miRNAs, 30 ng of total RNA was subjected to RT (reverse transcription) using a TaqMan® microRNA Reverse Transcription Kit (Applied Biosystems) and Megaplex RT primers (Applied Biosystems) following the manufacturer’s protocol. RT was performed on a Mastercycler Epgradient thermocycler (Eppendorf) with the following cycling conditions: 40 cycles at 16 °C for 2 min, 42 °C for 1 min and 50 °C for 1 s followed by a final step of 80 °C for 5 min to inactivate reverse transcriptase. The expression profile of miRNAs was determined using the TaqMan® Universal Master Mix II (Life Technologies, Applied Biosystems) in an Applied Biosystems 7900HT thermal cycler using the manufacturer’s recommended program. Finally, all the raw data from each array were retrieved from the 7900HT and run on Data Assist Software ver.3.1 (Applied Biosystems).
4
Comprehensive miRNA Profiling by TLDA
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miRNA profiling was performed using TaqMan Low Density Arrays (TLDA) and pools A and B (Applied Biosystems, Waltham, MA, USA), which allow to analyze 768 miRNAs. Total RNA was retrotranscribed through the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and MegaPlex RT primers (Applied Biosystems) pools A and B. cDNAs were preamplified using TaqMan PreAmp Master Mix and PreAmp primers (pools A and B; Applied Biosystems). The miRNA arrays were loaded with the preamplified samples and run on a 7900HT Fast Real-Time PCR System (Applied Biosystems).
5
Quantitative miRNA Expression Analysis
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Megaplex™ RT Primers (Applied Biosystems), which are 380 stem-looped reverse transcripts that enable the synthesis of cDNA for mature miRNAs, were used. The TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) was used to make cDNAs for mature miRNAs. The SYBR Green Master Mix (Applied Biosystems) was used to amplify miR-377, using specific miR-377 primers [55 (link)] 5’-GAGCAGAGGTTGCCCTTG-3’ (forward) and 5’-ACAAAAGTTGCCTTTGTGTGA-3’ (reverse). The U6 small nuclear RNA primers[55 (link)] 5’-CTCGCTTCGGCAGCACA-3’ (forward) and 5’-AACGCTTCACGAATTTGCGT-3’ (reverse) were used as an internal control.
6
Reverse Transcription and miRNA Profiling
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Reverse Transcription (RT) reactions were prepared by pipetting 500–1,000 ng aliquots of total RNA from each embryonic organ of interest in 0.7 mL PCR tubes, and pipetting the reagents following the specifications for Megaplex RT Primers and TaqMan® MiRNA Reverse Transcription Kit (Applied Biosystems). 6.0 μL aliquots of each RT reaction were pipetted into 1.7 mL tubes containing 450 μL of TaqMan Universal PCR Master Mix (Applied Biosystems) and 444 μL of RNase-free water, mixed by vortexing, and pipetted into microfluidic TaqMan® Rodent miRNA Array A Low-Density Arrays plates. PCR amplification was performed using an Applied Biosystems 7900HT Fast Real-Time System Thermocycler according to specifications described on MegaPlexTM Pools microRNA Expression Analysis brochure (Applied Biosystems).
7
Profiling Murine Immune Cell miRNAs
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Taqman low-density array (TLDA) was performed using TaqMan® Array Rodent MicroRNA A + B Cards Set v2.0 (Applied Biosystems) as previously described
[55 (link)]. Samples used were whole cell, cytoplasmic and nuclear RNA from LSK, promyelocytes, myelocytes and granulocytes. Briefly, reverse transcription reaction was performed using rodent Megaplex™ RT primers (Applied Biosystems), which contain a pool of 750 individual miRNA-specific primers, according to the manufacturer’s instructions. Real-time quantitative PCR (RT-qPCR) was then carried out on an ABI 7900HT real-time PCR machine with the LDA thermal cycler block, using pre-defined TLDA thermal cycling conditions. RT-qPCR data were analyzed using SDS 2.3 and RQ Manager Software (Applied Biosystems). The whole cell and cytoplasmic miRNA CT values correlated with R2 values of 0.9126 (LSK), 0.9112 (promyelocyte), 0.9244 (myelocyte) and 0.9406 (granulocyte). Raw data were deposited in the Gene Expression Omnibus database (accession number GSE57624).
[55 (link)]. Samples used were whole cell, cytoplasmic and nuclear RNA from LSK, promyelocytes, myelocytes and granulocytes. Briefly, reverse transcription reaction was performed using rodent Megaplex™ RT primers (Applied Biosystems), which contain a pool of 750 individual miRNA-specific primers, according to the manufacturer’s instructions. Real-time quantitative PCR (RT-qPCR) was then carried out on an ABI 7900HT real-time PCR machine with the LDA thermal cycler block, using pre-defined TLDA thermal cycling conditions. RT-qPCR data were analyzed using SDS 2.3 and RQ Manager Software (Applied Biosystems). The whole cell and cytoplasmic miRNA CT values correlated with R2 values of 0.9126 (LSK), 0.9112 (promyelocyte), 0.9244 (myelocyte) and 0.9406 (granulocyte). Raw data were deposited in the Gene Expression Omnibus database (accession number GSE57624).
8
Cadmium-induced miRNA Expression
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RNA was extracted using the MIRVANA kit (AMBION) and was reverse transcribed with Taqman MicroRNA Reverse Transcription Kit using Megaplex RT Primers (Applied Biosystems).
MicroRNA expression was measured and quantified using microfluidic cards (Taqman ArrayMicroRNA Cards, set A, V2.2 and set B, V3, Applied Biosystems) allowing the detection of about 754 unique assays specific and four candidate endogenous control assays (Applied Biosystems, Foster City, CA). Samples of control and 10 μM Cd were tested. Analyses were carried out on the ABI Prism 7900HT Sequence Detection System (SDS) (Applied Biosystems). MicroRNA levels were normalized to endogenous control U6 (Applied Biosystems). MiRNAs with a threshold cycle <33 that showed a log2 fold change greater than two in samples treated with cadmium as compared to control samples were considered as induced.
MicroRNA expression was measured and quantified using microfluidic cards (Taqman ArrayMicroRNA Cards, set A, V2.2 and set B, V3, Applied Biosystems) allowing the detection of about 754 unique assays specific and four candidate endogenous control assays (Applied Biosystems, Foster City, CA). Samples of control and 10 μM Cd were tested. Analyses were carried out on the ABI Prism 7900HT Sequence Detection System (SDS) (Applied Biosystems). MicroRNA levels were normalized to endogenous control U6 (Applied Biosystems). MiRNAs with a threshold cycle <33 that showed a log2 fold change greater than two in samples treated with cadmium as compared to control samples were considered as induced.
9
MicroRNA Profiling for DLBCL Biomarkers
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The first step of this study was the selection of the microRNAs that will be
quantified in all the samples of the biobank and that could potentially be used
as biomarkers. To this end, 2 patients with DLBCL and 1 healthy donor with no
history of cancer were selected for a microRNA profiling. These patients were
selected based on their highly different response to treatment.
A profiling of 377 microRNAs was performed by TaqMan Low-Density Array (Applied
Biosystems, Life Technologies Europe BV, Merelbeke, Belgium) on 26 µL of total
RNA fraction extracted from patient samples. This volume of extracted RNA was
concentrated to dryness in a vacuum concentrator (Hetovac VR-1, Heto Lab) and
used for the reverse transcription step with the Taqman MicroRNA Reverse
Transcription Kit and the Megaplex RT Primers from Applied Biosystems, Human
Pool A v2.1. The quantification of 377 microRNAs was finally performed in a
TaqMan Array, Human MicroRNA A Card v2.0 using the ViiA 7 system (Applied
Biosystems, Life Technologies Europe BV, Merelbeke, Belgium). MicroRNAs with a
Cq higher than 32 were considered as nonexpressed. The relative expression of
microRNAs was calculated by the 2−ΔΔCq method using the U6-snRNA
level, selected by the NormFinder software, as an endogenous control for
normalization.
quantified in all the samples of the biobank and that could potentially be used
as biomarkers. To this end, 2 patients with DLBCL and 1 healthy donor with no
history of cancer were selected for a microRNA profiling. These patients were
selected based on their highly different response to treatment.
A profiling of 377 microRNAs was performed by TaqMan Low-Density Array (Applied
Biosystems, Life Technologies Europe BV, Merelbeke, Belgium) on 26 µL of total
RNA fraction extracted from patient samples. This volume of extracted RNA was
concentrated to dryness in a vacuum concentrator (Hetovac VR-1, Heto Lab) and
used for the reverse transcription step with the Taqman MicroRNA Reverse
Transcription Kit and the Megaplex RT Primers from Applied Biosystems, Human
Pool A v2.1. The quantification of 377 microRNAs was finally performed in a
TaqMan Array, Human MicroRNA A Card v2.0 using the ViiA 7 system (Applied
Biosystems, Life Technologies Europe BV, Merelbeke, Belgium). MicroRNAs with a
Cq higher than 32 were considered as nonexpressed. The relative expression of
microRNAs was calculated by the 2−ΔΔCq method using the U6-snRNA
level, selected by the NormFinder software, as an endogenous control for
normalization.
10
Quantifying miRNA Profiles via RT-PCR
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After establishing RNA purity (OD ratio of 260/280), the samples were subjected to reverse transcription using MegaPlex RT Primers and TaqMan miRNA reverse transcription kits (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. The cDNA was diluted in Universal PCR master mix-II (Applied Biosystems) and then loaded on to TaqMan® Low Density Array (TLDA) microfluidic MicroRNA 384-well cards (Applied Biosystems) for real-time PCR (ABI 7900 HT RT-PCR System). The relative concentration of miRNAs was calculated by comparative (RQ = 2−ΔΔCt) analysis and the fold change determined as log10 RQ. The data were analyzed using RQ Manager 12.1 (Applied Biosystems) and RealTime StatMiner software (Integromics, Philadelphia, PA, USA).
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