mRNA from cells was isolated using RNeasy Mini or Micro Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. RNA from tissue was extracted using TriPure Isolation Reagent (Sigma–Aldrich). RNA was synthesized to cDNA using a mix of Oligo (dT) 12–18 Primer, random primers, dNTP mix (100 mM), and SuperScript™ III Reverse Transcriptase, all from Invitrogen. qPCR was performed on a CFX96™ Real‐Time PCR Detection System, using Platinum™ SYBR™ Green qPCR SuperMix‐UDG w/ROX (Thermo Fisher), 10 μM of primers, and 1 μl of cDNA (5 ng/μl). qPCR data were retrieved and processed using the CFX Manager™ software (Bio‐Rad, USA). All primers used are specified in Table S1 .
Dntp mix 100 mm
Manufactured by Thermo Fisher Scientific
DNTP mix (100 mM) is a laboratory reagent that provides a pre-mixed solution of the four deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) in an aqueous buffer. Each deoxynucleotide is present at a concentration of 100 mM.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
Oligo dt 12 18 primer, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini, by Qiagen (1 mentions)
Tripure isolation reagent, by Merck Group (1 mentions)
Platinum sybr green qpcr supermix udg w rox, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
G box, by Syngene (1 mentions)
Omniscript rt kit, by Qiagen (1 mentions)
2 protocols using dntp mix 100 mm
1
qPCR of mRNA Extraction and Synthesis
2
Extraction and RT-PCR Analysis of Total RNA
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Total RNA was isolated from HEL cells with the RNeasy® Mini Kit following the RNA extraction and semi-quantitative RT-PCR manufacturer's protocol (Qiagen). 2 µg total RNA were retro-transcribed into cDNA using the first-strand cDNA synthesis part of Omniscript RT Kit (Qiagen), oligo-dT (25 µg) (Invitrogen, Cergy Pontoise, France) and RNase Out™ (40 U/ml) (Invitrogen). 2 µl of reverse-transcribed cDNA was used for PCR according to the Kit HotStar Taq DNA Polymerase (250 units) protocol (Qiagen) with dNTP mix (100 mM) (Invitrogen) and 0.5 µM of sense and antisense primers. The primers for PCR were chosen to amplify human GpV and we used GAPDH as an internal control. PCR resulting fragments were visualized as previously described (21 (link)) by the GBOX (Syngene) and the Genesnap version 7.09 software. Bands quantification was done by Image J software.
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