For qRT-PCR, cells were growing on 6-well plate. After treatment with the corresponding drug, total RNA was isolated using Universal RNA/miRNA Purification Kit (EURx, Poland). RNA was quantified using Nanodrop ND-1000 (ThermoScientific, USA). cDNA was obtained using LunaScript™ RT SuperMix Kit (New England Biolabs, USA) and qPCR reactions were performed using Luna® Universal qPCR Master Mix (New England Biolabs, USA) according to manufactural instructions. The qPCR reactions were done in CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA) and then analyzed in CFX Maestro Analysis Software (Bio-Rad, USA). The final graphs were prepared in GraphPad Prism 8. Primers for amplification spanned the A’/01 site, ensuring that only 47S and no other precursors were amplified. The sequences were: GTGCGTGTCAGGCGTTC and GGGAGAGGAGCAGACGAG.
Universal rna mirna purification kit
Manufactured by EURx
Sourced in Poland
The Universal RNA/miRNA Purification Kit is a laboratory tool designed to isolate and purify total RNA, including microRNA (miRNA), from a variety of biological samples such as cells, tissues, and body fluids. The kit utilizes a silica-based membrane technology to efficiently capture and elute RNA molecules of all sizes.
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Lab products found in correlation
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Cfx maestro analysis software, by Bio-Rad (1 mentions)
Lunascript rt supermix kit, by New England Biolabs (1 mentions)
Luna universal qpcr master mix, by New England Biolabs (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Ng dart rt pcr kit, by EURx (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
M mlv reverse transcription kit, by Promega (1 mentions)
Thermal cycler uno96, by Avantor (1 mentions)
Mircury lna rt kit, by Qiagen (1 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Mircury sybr green pcr kit, by Qiagen (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Experion automated electrophoresis system, by Bio-Rad (1 mentions)
Fta classic card, by Cytiva (1 mentions)
Rl buffer, by EURx (1 mentions)
Whatman, by Cytiva (1 mentions)
7 protocols using universal rna mirna purification kit
1
Quantitative RT-PCR Analysis of 47S Precursor
2
Comparative qPCR analysis of gene expression
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RNA from HUVEC cells or blood cells or monocytes were isolated with Universal RNA/miRNA Purification Kit (EURx) according to the manufacturer’s protocol. cDNAs were generated with NG dART RT-PCR kit (EURx) or High-Capacity cDNA Reverse Transcription Kit (ThermoFischer) following the manufacturer’s protocol. Comparative qPCR was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems) with PerfeCTa SYBR Green FastMix, Low ROX (Quantabio). For comparative qPCR, 40 cycles were used with initial denaturation at 95 °C for 10 min, denaturation at 95 °C for 15 s, annealing, extension, and reading fluorescence at 60 °C for 1 min. Human ACTB was used as housekeeping gene for relative quantification in human blood cell or monocyte qPCR, LGALS1 was used for HUVEC qPCR, and murine ACTB was used for mouse blood cell qPCR. Primers used for the study are listed in Supplementary Table 1 . qPCR data were captured and analyzed using StepOne v2.3.
3
RNA Isolation and cDNA Synthesis
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RNA was isolated using the Universal RNA/miRNA Purification Kit (Eurx), according to vendor’s protocol. RNA was reverse transcribed to cDNA using the Moloney Murine Leukemia Virus reverse transcription kit (M-MLV Reverse Transcriptase, Promega, Madison, WI, USA), according to vendor’s protocol.
4
Gene Expression Analysis by RT-PCR
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The gene expression was analyzed at mRNA level by RT-PCR. Total RNA was isolated by Universal RNA/miRNA Purification Kit (Eurx), according to the manufacturer’s instruction. After measurement of RNA concentration, reaction of reverse transcription (RT) was performed with M-MLV reverse transcription kit (Promega, Madison, WI, USA).
5
Barley miRNA Expression Analysis
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Two-step quantitative real-time PCR (qRT-PCR) analysis was performed to assess miRNA expression levels in barley varieties “Marthe” and “KWS Olof” grown with different concentrations of Fe3O4 and CuO NPs compared to control plants. miRNAs were extracted from the shoots using a Universal RNA/miRNA Purification Kit (EURx, Poland). RNA was extracted from 140 samples from “Marthe” and “KWS Olof” variety fresh shoots (20 in each experimental group). RNA was quantified and qualified with a spectrophotometer (NanoDrop One, Thermo Scientific, USA). Samples with an A260/280 ratio from 1.7 to 2.1 were used for analysis. miRNA target-specific primer hvu-miR156a with locked nucleic acid was designed. The target miRNA hvu-miR156a sequence was 5′-TGACAGAAGAGAGTGAGCACA-3′. HvsnoR14 was used as a reference gene for the normalization of miRNA expression values. Reverse transcription for miRNAs was performed using the Thermal Cycler UNO96 (VWR, United Kingdom) and miRCURY LNA RT Kit (Qiagen, Germany) according to the protocol first-strand cDNA synthesis. For miRNA qRT-PCR analysis, miRCURY SYBR Green PCR kit (Qiagen, Germany) was used according the manufacturer's protocol. The Rotor Gene Q Series Software program was used to analyse the miR156a expression level of barley varieties. The results were analysed using the 2 − ΔΔCT method [40 (link)].
6
RNA Extraction Using Universal Kit
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RNA extraction was performed using a Universal RNA/miRNA purification kit (EURX, Gdańsk, Poland) according to the manufacturer’s instructions. RNA was eluted using 50 µL of RNAse-free water. The RNA concentration was estimated using NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, MA, USA), and RNA integrity was confirmed using an Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA). In all cases, one biological replicate was pooled from six independent plants.
7
FTA Card RNA Isolation Protocol
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Blood samples collected on FTA Classic Cards (Whatman) from 10 patients were used for RNA isolation using the Universal RNA/miRNA Purification Kit (EURx). FTA card pieces, approximately 0.5 cm 2 in size, were incubated in 400 µl of RL buffer (EURx) at room temperature for 30 minutes. Next, a 100 µl portion of the Lyse ALL buffer (EURx) was added, and the mixture was centrifuged at 12,000 rcf for two minutes. The supernatant was transferred into a homogenization column and treated with DNAse. RNA was extracted according to the protocol specified by the kit manufacturer. RNA was suspended in 50 µl of distilled water free from RNAses and DNAses (Gibco).
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