Three PCR-generated fragments containing the miR-30a binding sites (one in NOTCH1, two in NOTCH2) (Supplementary Figure 1 ) were individually cloned in the 3′UTR of the luciferease gene (pMIR vector, Ambion). Site directed mutagenesis was used to modify three of the six “seed sequence” nucleotides in each construct, as we described23 (link). After sequencing verification, these constructs were cotransfected in HEK-293 cells with the pCMVβ-gal plasmid (5ng) and 100 nM each of chemically synthesized pre–miR-30a or pre–miR negative control oligonucleotides (Sigma). Cells were harvested 48 h after transfection, and luciferase and β-galactosidase activities measured, as we described24 (link). The primers sequences are listed in Supplementary Table 3 .
Pre mir 30a
Manufactured by Merck Group
Pre–miR-30a is a synthetic precursor molecule designed to mimic the natural miRNA-30a. It is used in research settings to study the biological functions and regulatory mechanisms of miRNA-30a.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pre mir 30a
1
Validation of miR-30a Binding Sites
2
Validation of miR-30a Binding Sites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three PCR-generated fragments containing the miR-30a binding sites (one in NOTCH1, two in NOTCH2) (Supplementary Figure 1 ) were individually cloned in the 3′UTR of the luciferease gene (pMIR vector, Ambion). Site directed mutagenesis was used to modify three of the six “seed sequence” nucleotides in each construct, as we described23 (link). After sequencing verification, these constructs were cotransfected in HEK-293 cells with the pCMVβ-gal plasmid (5ng) and 100 nM each of chemically synthesized pre–miR-30a or pre–miR negative control oligonucleotides (Sigma). Cells were harvested 48 h after transfection, and luciferase and β-galactosidase activities measured, as we described24 (link). The primers sequences are listed in Supplementary Table 3 .
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!