MicroRNA (miRNA) functional analyses can be performed by using miRNA inhibitors (Anti-miR™ miRNA Inhibitors) and synthetic miRNA mimics (Pre-miR™ siRNA Precursors), which are now available for most known miRNAs. Anti-miR and Pre-miR positive, negative, and labeled controls have been designed to serve as convenient and effective controls for experiments involving Anti-miR miRNA Inhibitors and Pre-miR miRNA Precursors.

Positive Control: Pre-miR™ hsa-miR-1 miRNA Precursor

Until now, there has been no known miRNA positive control with robust and confirmed functional effects on gene expression in cultured mammalian cells. To address this need, Ambion scientists have designed a Pre-miR hsa-miR-1 miRNA Precursor, a double-stranded RNA oligonucleotide, that can be used as a positive control in experiments along with other Pre-miR miRNA Precursors.

Pre-miR hsa-miR-1 miRNA Precursor, which mimics mature miR-1, has been shown to down-regulate the Protein Tyrosine Kinase 9 (PTK9; twinfillin) gene at the mRNA level [1]. Expression of PTK9 mRNA is decreased by 60–95% following Pre-miR hsa-miR-1 miRNA Precursor transfection, compared to transfection with Pre-miR miRNA Precursor–Negative Control #1, using real-time PCR and TaqMan® Gene Expression Assays [Assay ID#s Hs00702289_s1 (human) and Mm01598980_g1 (mouse); data not shown]. Therefore, Pre-miR hsa-miR-1 miRNA provides a convenient positive control for miRNA-mediated gene silencing of PTK9, when monitored using the appropriate TaqMan Gene Expression Assays and real-time PCR. The Pre-miR positive control can be used to confirm that the transfection procedure in cell cultures support miRNA-mediated gene silencing.

Anti-miR™ and Pre-miR™ miRNA Negative Controls

Samples transfected with the Anti-miR and Pre-miR Negative Controls are used as a baseline for evaluating the effect of experimental Anti-miR miRNA Inhibitors and Pre-miR miRNA Precursors on target gene expression. Anti-miR and Pre-miR Negative Controls have been extensively validated in human cell lines and tissues and do not produce identifiable effects on known miRNA function.

Anti-miR miRNA Inhibitor–Negative Control #1 is a nontargeting sequence Anti-miR molecule designed to serve as a negative control for experiments involving Anti-miR miRNA Inhibitors. These Anti-miR miRNA negative controls are transfected using the same methods as those for experimental Anti-miR miRNA Inhibitors.


Pre-miR miRNA Precursor–Negative Control #1 and #2 are double-stranded RNA oligonucleotides with nontargeting sequences, which are designed to serve as negative controls for experiments involving Pre-miR miRNA Precursors.

Labeled Anti-miR and Pre-miR Negative Controls

Labeled Anti-miR and Pre-miR Negative Controls include FAM™ or Cy™3 fluorescent dyes on the 5'-end of the oligonucleotide. These fluorescently labeled molecules are useful in monitoring uptake in transfection experiments involving Anti-miR miRNA Inhibitors and Pre-miR miRNA Precursors. The labeled Anti-miR negative control consists of oligonucleotides that have the same sequence as Anti-miR miRNA Inhibitor–Negative Control #1, and the labeled Pre-miR negative controls consists of oligonucleotides that have the same sequence as Pre-miR miRNA Precursor–Negative Control #1. Their most common application is to monitor transfection efficiency during optimization of transfection conditions. 

References

1. Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs, Nature 433(7027):769–773.