Luciferase reporter vectors were constructed by cloning miR-4261 and miR-298 into pcDNA6.2 vector (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA), 3′-untranslated region (UTR) of ZNF207 and 3′-UTR of ILF3 into pGL3 vector (Invitrogen), ZNF207 and ILF3 into pcDNA3.1 vector (Invitrogen), and promoter of EZH2 into pGL3 vector (Invitrogen). SKOV3 cells were cotransfected with pcDNA6.2 or pcDNA3.1 vector and pGL3 vector by Lipofectamine 2000. After 48 hours, luciferase assays were performed using the dual luciferase reporter assay system (Promega Corporation, Fitchburg, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. All assays were performed in triplicate.
Pcdna6.2 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA6.2 vector is a plasmid designed for the expression of recombinant proteins in mammalian cell lines. It contains a cytomegalovirus (CMV) promoter for high-level expression and a blasticidin resistance gene for selection of stable transfectants.
Automatically generated - may contain errors
Lab products found in correlation
Pgreenpuro vector, by System Biosciences (3 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (2 mentions)
Pgl3 vector, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Cignal 45 pathway reporter array, by Qiagen (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
2 protocols using pcdna6.2 vector
1
Luciferase Reporter Assay for miRNA Targets
2
Constructing Vectors for miRNA and Target Studies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the stable miRNA expressing vector, an approximately 300 bp DNA fragment containing pre‐miR‐372 or pre‐miR‐373 was amplified from SW480 genomic DNA and cloned into the pISNCG vector. For miRNA transient overexpression vectors, the 300 bp DNA fragment containing the entire pre‐miR‐372 or pre‐miR‐373 sequence was cloned into the pcDNA6.2 vector (K4936‐00; Invitrogen). For the stable miR‐TuD‐expressing vector, the 146 nt TuD stem loop sequence, which could sequester miR‐372/373, was synthesized and then cloned into the pGreen‐Puro vector (System Bioscience).
For target validation, both strands of the 59 nucleotide 3′ UTR sequence containing the miRNA binding region of each target gene were synthesized, directly annealed and then cloned into the psiCheck2 vector (Promega, Madison, WI, USA).
For gene overexpression vectors, the RNA was reverse transcribed and fragments containing the VDR, SPOP and SETD7 were amplified and cloned into the pcDNA3.1 vector (V790‐20; Invitrogen).
Cignal 45‐pathway reporter arrays that measure the activity of the 45 pathway were purchased from Qiagen (Hilden, Germany).
Primers for vector construction are listed inTable S2 .
For target validation, both strands of the 59 nucleotide 3′ UTR sequence containing the miRNA binding region of each target gene were synthesized, directly annealed and then cloned into the psiCheck2 vector (Promega, Madison, WI, USA).
For gene overexpression vectors, the RNA was reverse transcribed and fragments containing the VDR, SPOP and SETD7 were amplified and cloned into the pcDNA3.1 vector (V790‐20; Invitrogen).
Cignal 45‐pathway reporter arrays that measure the activity of the 45 pathway were purchased from Qiagen (Hilden, Germany).
Primers for vector construction are listed in
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!