The Ultimate in Functional miRNA Target Identification

The MISSION Target ID Library enables bench-top transcriptome-wide human miRNA and ncRNA gene target identification. With an innovative dual-positive selection system, it makes rapid whole transcriptome miRNA and ncRNA gene target screens available to any researcher with minimal time, reagent, or capital equipment expense.

• Features and Benefits
• Product Description
• Workflow
• Vector Map

MISSION Target ID Library Benefits

• Convenience: Perform dual screening with one vector due to the innovative dual selection cassette
• Simplicity: 3 easy steps:
  • Library Transfection
  • microRNA Transfection and Ganciclovir Selection
  • PCR and Sequencing
• Relevance: The only method for genome-wide functional identification of miRNA targets

Product Description

Experimentally Identify miRNA targets by screening the human transcriptome for functional binding sites and transcriptional regulation of these targets

The MISSION Target ID Library is a pool of plasmids, each with a single human cDNA inserted into the 3′-UTR of a thymidine kinase-zeocin fusion protein (TKzeo). With dual-selection, the user will be able to transfect cells to generate a stable cell line expressing the library and then screen the entire library at once for miRNA targets. The first selection selects for stable transformants and the second, after introducing a miRNA or ncRNA of interest, selects for surviving cells containing the miRNA target cDNAs. The target cDNAs are then identified by PCR and sequencing.

The human cDNA library used to create the MISSION Target ID library is produced from several different combined pools of total human RNA. The average length of the cDNA in the library is 1.2kb; the library was oligo dT primed (starting at the poly A tail of the mRNA) and includes all of the 3’UTR and some coding region for most genes. Each library is tested for gene representation to ensure robust library coverage. The library is provided in ready-to-use DNA format, with enough material to create at least 6 cell lines with a stably integrated library for target identification.

MISSION Target ID Library Workflow

MISSION Target ID Library Transfection

Cells are transfected with the MISSION Target ID Library and allowed to recover for 3-5 days. Constructs can integrate into the genome during this recovery period and express the encoded transcript.

Zeocin Selection (Positive Selection)

After recovery, cells are exposed to Zeocin. Zeocin is an antibiotic and only cells expressing a Zeocin resistant gene will survive. Therefore, cells expressing the TKzeo fusion protein from stably integrated Target ID constructs survive. Untransfected cells die.

Any cells containing a construct that is a target for an endogenous miRNA will also block expression of the TKzeo gene and these cells will also die. This is designed to reduce false positives in the final selection step due to endogenous miRNA expression in the cell.

Ideally, the miRNA of interest will not have a high expression level in the cell line, as these gene targets will be removed during positive selection.

microRNA Transfection

Cells containing the MISSION Target ID Library are transfected with a selectable miRNA expression construct. During recovery, the miRNA expression construct can integrate and express the encoded selectable marker.

Ganciclovir Selection (Negative Selection)

After selection for stable integration and cell expansion, cells are treated with Ganciclovir.

Cells producing HSV thymidine kinase (HSVTK) in the presence of Ganciclovir will die. These are cells expressing TKzeo constructs not targeted by the miRNA.

Cells containing TKzeo constructs with miRNA target sites will not produce HSVTK and will survive Ganciclovir selection. These cells contain the miRNA target gene constructs.

PCR and Sequencing

To identify the targets, genomic DNA is isolated and PCR is performed with primers provided. These primers flank the cDNA insert sites and only inserts are amplified. The PCR products can be cloned and sequenced or deep sequencing can be performed for identification.

Vector Map