To extract the miRNAs from each sample, a high purity miRNA isolation Kit (Roche) was used. The solutions were quantified using the Qubit® 2.0 Fluorometer (Invitrogen) and diluted to a final concentration of 4 ng/μL. Then, the cDNA was obtained using TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies), and the Quantitative Real-Time PCR reaction was performed using the TaqMan® MicroRNA Assays in a 7500 Real Time PCR system (Life Technologies) with TaqMan miRNA assays according to the manufacturer’s instructions (Life Technologies) using primers designed by Primer Express (Life Technologies). The mean expression level of three human endogenous controls (Z30, RNU19 and RNU6B – calibrators) was used as an internal control in all of the miRNA experiments to allow the comparison of expression results. The relative miRNA expression levels were then calculated by a comparative threshold cycle (Ct) method (2−Δct).
Mirna isolation kit
Manufactured by Roche
Sourced in Germany
The MiRNA isolation kit is a laboratory tool designed to extract and purify microRNA (miRNA) molecules from biological samples. It provides a standardized and efficient method for isolating miRNA from various sources, such as cells, tissues, or bodily fluids. The kit utilizes a proprietary technology to selectively capture and concentrate miRNA, allowing for its downstream analysis and applications.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Primer express, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Am7020, by Thermo Fisher Scientific (1 mentions)
4 protocols using mirna isolation kit
1
Quantitative Analysis of miRNA
2
Plasma miRNA Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For miRNA analysis, the samples were centrifuged at 2,000 g for 10 min without waiting for more than 2 h at room temperature. The aliquoted plasma samples were stored at −80°C in a freezer. MiRNA isolation was performed from plasma samples using a miRNA isolation kit (Roche Diagnostics, GmbH, Mannheim, Germany). After isolation, the samples were converted to cDNA and stored at −20°C until the analysis. The expression analysis of the 10 target miRNAs was performed using a real-time polymerase chain reaction (RT-PCR) device (Roche LightCycler 480). The Ct values of the miRNAs were normalized using the reference RNU6B. The fold change of miRNA expression for each miRNA was calculated as the 2-ΔΔCt method6 (link)
.
.
3
Extracting miRNA from FFPE Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MiRNA samples were isolated from Formalin-Fixed Paraffin-Embedded Tissues using the Roche miRNA isolation kit. Briefly, the paraffin specimens were cut into 5μm thick slices and then treated with xylene for 5 minutes at room temperature, then RNA the precipitated using pure ethonal. MiRNA samples were further isolated using the RNA binding column following the manufacturer's instructions.
4
Isolation and Quantification of mRNA and miRNA from MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of mRNA was performed from MSCs which had been exposed to 0.5 µg/ml S100A8/A9 for 6 and 24 h, respectively. Thereafter, MSCs were flushed with 10 ml PBS and scraped from cell culture plates on ice. Cell pellets were incubated with 100 µl of RNA later (Ambion, Cat # AM7020). Total RNA was extracted from MSC using Qiagen reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions and stored at −80 °C until further analysis. RNA quality was verified with Bioanalyzer and RNA samples were considered for further analysis when showing RIN number between 9 and 10. Control mRNA samples were isolated from non-treated MSCs. The miRNA was isolated with miRNA isolation kit (Roche, Cat #05080576001) as suggested by the manufacturer´s instructions using a 2 step isolation protocol. The miRNA quantification was carried out using Qubit 3.0 Fluorometer and Quant-IT miRNA assay kit (Thermofisher Scientific, Cat # Q32882).
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!