The RNA extraction was achieved using TRIzol (Life Technologies) according to the manufacturers protocol. 0.5 µg of RNA was reversely transcribed into cDNA using an NZY First-Strand cDNA Synthesis Kit (NZYTech, Lisbon, Portugal) following the manufacturer´s instructions. The RT-PCR was performed in a LightCycler 480-II Instrument (Roche, Mannheim, Germany) using TaqMan Universal Master Mix (Roche). Analysis of the results was carried out using Qbase+ version 2.5 software (Biogazelle, Ghent, Belgium). Gene expression was calculated relative to the housekeeping gene ribosomal protein L13A (RPL13A). Sequence primers, probe, and PCR conditions are available upon request.
Qbase version 2
Manufactured by CellCarta
Sourced in Belgium
Qbase+ version 2.5 is a software product that provides a core function for data analysis and management. The software is designed to handle various types of laboratory data.
Automatically generated - may contain errors
Lab products found in correlation
Taqman universal master mix, by Roche (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 instrument 2, by Roche (1 mentions)
Nzy first strand cdna synthesis kit, by NZYTech (1 mentions)
Superscript vilo, by Thermo Fisher Scientific (1 mentions)
Trizol, by Merck Group (1 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using qbase version 2
1
RNA Extraction and RT-PCR Analysis
2
RT-PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA extraction was achieved using TRIzol® (Sigma-Aldrich), following the manufacturer’s protocol. A total of 0.5 mg of RNA was reverse transcribed into cDNA using Super Script VILO (Thermofisher Scientific, Waltham, MA, USA), following the manufacturer´s instructions. RT-PCR was developed in a LightCycler 480-II Instrument (Roche, Mannheim, Germany) using TaqMan Universal Master Mix (Roche). The analysis of the results was carried out using Qbase+ version 2.5 software (Biogazelle, Ghent, Belgium). Gene expression was calculated relative to the housekeeping gene Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). Sequence primers (Table 3 ) and PCR conditions were described; a pre-incubation step at 95 °C for 5 min, followed by 40 cycles corresponding to amplification (denaturation at 95 °C for 10 seconds (s), annealing all the primers at 60 °C for 10 s with an extension at 72 °C for 1 s), and the last step was cooling at 4 °C for 5 min. Only a single acquisition mode was applied during the extension step in the amplification process.
3
Total RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from cells was extracted using Trizol® Reagent (Thermo Fisher Scientific) following the manufacture's procedures. Afterwards, 1μg of total RNA was reverse transcribed using NZY First-Strand cDNA Kit (NZY Tech Genes and Enzymes, Portugal) according to manufacturer's instructions.
Quantitative real-time PCR (qRT-PCR) reactions were performed in LightCycler 480 II instrument (Roche Molecular Biochemicals) using TaqMan Universal Master Mix (Applied Biosystems) Analysis of the results was carried out using Qbase+ version 2.5 software (Biogazelle, Gent, Belgium) using ∆∆ Ct method. Normalization was carried out considering as reference housekeeping gene ribosomal protein L13a (RPL13A). This gene was chosen by software geNorm version 3.5 (Primer design, Southampton University's School of Medicine, United Kingdom), an algorithm able to evaluate the most stable housekeeping gene derived from a group of tested candidate reference genes. The software determines the gene expression stability measure for a reference gene calculating the average pairwise variation of that gene in comparison to all other tested genes. Stepwise exclusion of the gene with the highest stability measure value allows to calculate the tested genes according to their expression stability [30] . Sequence of primers and probes are listed in Table 1.
Quantitative real-time PCR (qRT-PCR) reactions were performed in LightCycler 480 II instrument (Roche Molecular Biochemicals) using TaqMan Universal Master Mix (Applied Biosystems) Analysis of the results was carried out using Qbase+ version 2.5 software (Biogazelle, Gent, Belgium) using ∆∆ Ct method. Normalization was carried out considering as reference housekeeping gene ribosomal protein L13a (RPL13A). This gene was chosen by software geNorm version 3.5 (Primer design, Southampton University's School of Medicine, United Kingdom), an algorithm able to evaluate the most stable housekeeping gene derived from a group of tested candidate reference genes. The software determines the gene expression stability measure for a reference gene calculating the average pairwise variation of that gene in comparison to all other tested genes. Stepwise exclusion of the gene with the highest stability measure value allows to calculate the tested genes according to their expression stability [30] . Sequence of primers and probes are listed in Table 1.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!