Viral RNA was extracted from samples collected from test surfaces using the MagaZorb® total RNA Mini-Prep Kits (Promega, Madison, WI) according to the manufacturer’s instructions. One hundred μL samples were mixed with 250 μL of lysis buffer and 85 μL of 100% isopropanol. The resulting solution mixture was vortexed for 5 seconds and the lysate was transferred to a Minicolumn and centrifuged at 14,000 x g for 30 seconds, followed by a first wash with 500 μL of RNA wash solution, centrifuged at 14,000 x g for 30 seconds and then a second wash with 300 μL of RNA wash solution and centrifuged at 14,000 x g for 2 minutes. In the final step, 100 μL samples were used and nucleic acids were eluted with 50 μL of elution buffer. The RNA extracts were stored at -80°C until further analysis.
Magazorb total rna mini prep kit
Manufactured by Promega
Sourced in United States
The MagaZorb® Total RNA Mini-Prep Kit is a laboratory product designed for the rapid and efficient isolation of total RNA from a variety of sample types. The kit utilizes a proprietary magnetic bead-based technology to capture, wash, and elute high-quality RNA suitable for downstream applications such as RT-PCR, qRT-PCR, and other RNA-based analyses.
Automatically generated - may contain errors
Lab products found in correlation
Rt pcr kit, by Promega (2 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Nah2po4, by Merck Group (1 mentions)
Na2hpo4, by Merck Group (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Tris base, by Merck Group (1 mentions)
Bromphenol blue, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (1 mentions)
Nanophotometer, by Implen (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using magazorb total rna mini prep kit
1
Viral RNA Extraction from Surfaces
2
hCMV RNA Quantification in Cell Lines
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A 10-fold serial dilution of VR1814 from 1 to 104 IU/well was used to inoculate MRC-5, ARPE-19, and HMEC-1 cells in 96-well plates. After a 20-h incubation, cells were washed once with 200 μL per well of PBS and either extracted by MagaZorb® Total RNA Mini-Prep Kits (Promega, Madison, WI, USA) with a final volume of 50 μL elute in nuclease-free water or processed by the crude cell lysate as previously described by Shatzkes et al. with some modifications [20 (link)]. Briefly, 100 μL of buffer containing 10 mM Tris-HCl, pH 7.4, 0.25% Igepal CA-630 and 150 mM NaCl was added to each well for 10 min at room temperature followed by a vortex on a medium setting (setting 6 out of 8) for 30 s and a 15,000× g centrifugation at 20 °C for 2 min to obtain supernatants. hCMV RNA load in the extracted nucleic acid and crude cell lysate supernatant was analyzed by RT-qPCR in triplicate.
3
Protein Expression in E. coli
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Dulbecco’s modified eagles medium (DMEM, Gibco, USA), fetal bovine serum (Gibco, USA), penicillin and streptomycin (Sigma, USA), gentamycin (Sigma, USA), dimethylsulfoxide (Merck, Germany), trypsin (Merck, Germany), EDTA (Merck, Germany), Na2HPO4 (Merck, Germany), NaH2PO4 (Merck, Germany), isopropyl thiogalactopyranoside (IPTG, Sigma, USA) MTT (Merck, Germany), Tris base, (Merck, Germany), acetic acid (Merck, Germany), Ni TED column (Protino, USA), Kalferon (Kalbe farma, Indonesia), propidium iodide (Invitrogen, USA), MagaZorb® Total RNA Mini-Prep Kit (Promega, USA), RT-PCR kit (Promega, USA), PCR kit (Promega USA), primers : p27 (Forward and Reverse), casp3 (Forward and Reverse), GAPDH (Forward and Reverse) (First Base, Singapore), agarose (Boehringer Mannheim, Germany), DNA ladder 1 kpb (Fermentas, USA), sucrose (Sigma, USA), bromphenol blue (Sigma, USA) and ethidium bromida (Sigma, USA).
4
RNA Extraction from Inflorescence Apices
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Young inflorescences that only consist of inflorescence shoot apical meristem and buds at stages 1 to 5 were collected from lines APL01 and PL01, and three biological replicates were performed for each line. Then, total RNA was isolated using MagaZorb® Total RNA Mini-Prep Kit (Promega, Madison, USA). RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using a Qubit RNA Assay Kit on a Qubit 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Only pure RNA samples of high quality were used in RNA-seq.
5
Quantitative Real-Time PCR for Gli1 and AQP1
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Total RNA was extracted from normal tissues and glioma cells using MagaZorbⓇTotal RNA Mini-Prep Kit (Promega), according to the manufacturer’s protocols. RNA was quantified using spectrophotometer. Equal amount of RNAs was reversed using RETROscript kit (Ambion, Life Technologies Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocols. To evaluate quantitative changes in mRNA levels of Gli1 and AQP1, each RT reaction mixture was then subjected to real time PCR with Brilliant III Ultra-Fast SYBRH Green QPCR Master Mix (Stratagene, La Jolla, CA, USA). The PCR primers for detecting specific transcripts were as follows: Gli1, 5’-GCCAGCCAAGAGAGACCAACAG-3’ and 5’-CCGACAGAGGTGAGATGGACAG-3’; AQP1, 5’-GAAGAAGCTCTTCTGGAGGGC-3’ and 5’-AGCCAGGTCATTGCGGCCAAG-3’; β-actin, 5’-ACGTTGCTATCCAGGCTGTCCAT-3’ and 5’-TTAATGTCACGCACGATTTCCCGC-3’. Relative mRNA concentrations were calculated from the take-off point of reactions (threshold cycle, Ct) using the comparative quantitation method based upon the 2DDCt method. Expression of β-actin was considered as internal reference.
6
RT-PCR Analysis of Gene Expression
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Total RNA isolated from treated cells was used as a template. cDNA was synthesized from the same concentration of total RNA by using specific primers i.e. GAPDH, p27KIP, p21V1, p21B and caspase 3 (Table 1 ). cDNA amplification products were characterized by agarose gel electrophoresis and stained with ethidium bromide. The intensity of the bands was measured by ImageJ software to obtain semi-quantitative analysis. GAPDH band was used as internal control. Treated cells in 6-well plate were washed with PBS and detached with trypsin-EDTA. The cell suspension was centrifuged at 500 g and the supernatant was discarded. Total RNA was isolated from cells by using MagaZorb® Total RNA Mini-Prep Kit (Promega, USA) and the concentration was measured at 280 nm by spectrophotometer UV/Vis (Gene quant, USA). RT PCR was performed for each gene by RT-PCR kit (Promega, USA) at 0.5 µg of total RNA, 200 μM of dNTPs, 1.5 mM of MgCl2, 1U of Taq DNA polymerase and 2.5 µM of primer. cDNA amplification was carried out for each gene (Table 2 ).
7
Comprehensive DNA and RNA Extraction from Fresh Tissue
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A total of 206 DNA samples were extracted from fresh tissue which were collected from surgery or endoscopic biopsy and cryopreserved immediately in liquid nitrogen without formalin-fixed pretreatment using the MagaZorb® DNA Mini-Prep Kit (Promega, WI, USA), 149 RNA samples were extracted from fresh tissue using MagaZorb® Total RNA Mini-Prep Kit (Promega, WI, USA). Two experienced pathologists independently evaluated pathological features of hematoxylin and eosin-stained formalin-fixed, paraffin embedded (FFPE) tumor slides as well as confirmed at least 20% of the GC cell content of each sample.
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