Natural microRNA Precursor

Natural microRNA Precursor™

 

Natural microRNA precursors are microRNA transcripts that have not yet been processed into mature microRNA. These microRNA precursors can be delivered safely into the cell, where the precursors readily associate with microRNA processing complexes, and be processed into mature microRNAs.  These precursors are single stranded RNA molecules that contain the hairpin construct necessary to be assembled into RNA-induced silencing complexes (RISCs) for target gene silencing.  These molecules are the closest representation to the microRNA precursors found in cells.  Mature microRNA can then be used to study a multitude of cellular processes based on their ability to silence as well as indirectly upregulate various genes.  The advantage of using Natural microRNA precursors versus synthetic mimics is the ability to use the same microRNA precursors that are present naturally in the cellular environment.

 

Key Features

 

  • Natural: Natural microRNA precursors, not synthetic siRNA nor shRNA mimics
  • Quality: Microarray certified composition quality
  • Quantity: High quantity available for in-vivo research (HPLC)
  • Validated: Corresponding siRNA/shRNA mimics are validated by publications

 

Natural microRNA precursors from Mello Biotechnology are transcribed from a proprietary microRNA plasmid.  This plasmid has been sequenced to ensure correct sequence, and it is the only natural representation of cellular microRNA available on the market.  Traditionally, only synthetic mimics of microRNAs have been available, however it is unknown whether these microRNA mimics may interfere with exact function in natural cell state.  The clear advantage of using Natural microRNA precursor is the ability for the researcher to study the true role of microRNA in the cell without the worry of other variables that come with using altered forms of microRNA that is not naturally found in cells.  They are better able to represent the in vivo environment of an ESC, providing a system more closely related to nature than is possible using synthetic microRNA mimics.

Note: Mature microRNA sequence in red.

Why Natural?

 

Natural microRNA Precursor facilitates research of authentic microRNA function.  It does so by delivering natural microRNA function through a natural biogenesis mechanism.  Natural microRNA then functions via translational suppression, whereas synthetic shRNA or siRNA simply mimic this function via direct messenger RNA degradation.  Natural microRNA precursors provide researchers with the ability to eliminate other variables that can be introduced by using mimics.  Since our Natural miRNA precursor is a naturally occurring single-stranded form that contains the same hairpin constructs seen in natural cellular counterparts, and thus can perform the same functions. 

Our microRNA Precursor delivers its function through a pathway that proceeds naturally.  Natural microRNA Precursor may contain both unmatched sense and anti-sense microRNA that are not complimentary to each other, whereas synthetic siRNA and shRNA mimics have perfectly matching sense and anti-sense.  As a result, the anti-sense in synthetic mimics is different from natural anti-sense microRNA.  This difference often results in different targeting which can lead to unexpected off targeting effects.

[Natural microRNA vs Synthetic Pic]

[microRNA vs. siRNA biogenesis Pic]

Purified vs. Cell Extract

 

Natural microRNA precursors are provided in two forms: Cell Extract and Purified. Although both contain ample amount of precursors for experimental use, each version provide different benefits.  The raw extract form contains the hairpin constructs, as well as other total RNA molecules.  The plasmid, which contains a GFP reporter, can be used as a reporter to identify transfected cells.  In addition, total RNA protects microRNA precursors from degradation by RNAses, providing a more stable shelf life.  The Purified form is pure microRNA precursor, without any vectors or debris.  This ensures that only microRNA precursor is present, hence minimizing experimental variables.  However, removal of other RNA contents in cell extract may decrease the shelf-life of mircoRNA precursors, and increase their susceptibility to RNAse degradation

Quality & Stability

 

Natural microRNA precursors generated from specific microRNA plasmids undergo quality analysis to ensure sequence integrity.  These mircoRNA precursors contain the same sequence as naturally occurring cellular precursors and can therefore be used in experiments to learn about the direct effects of microRNA on cellular processes.  The precursors have been isolated from dicer-negative microRNA expressing competent cells that are capable of expressing ample amount of specific microRNA precursors.  Since cells are dicer-negative, they only produce miRNA precursors but not mature microRNAs.  Multiple purification processes are performed to ensure a high concentration of precursors in its pure state.

Single-stranded RNase first is introduced to digest any impurity RNAs.  Since microRNA precursors hairpins contain double-stranded regions, only undesired single stranded RNAs are removed.  The microRNA precursor size and purification is confirmed by electrophoresis.

Finally, the exact sequence and presence of the microRNA precursors are microarray certified to ensure that only pure population of specific natural microRNA precursors are present.


Electrophoresis confirms size & resistance to single-stranded RNase contamination


microArray data: Control & miR-302bcad Precursors

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Reference

  1. Lin et al., (2010) MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of CDK2 and CDK4/6 cell cycle pathways. Cancer Res., 70: 9473-9482. 
  2. Lin et al., (2011) Regulation of somatic cell reprogramming through inducible miR-302 expression.  Nucleic Acids Res., 39: 1054-1065. 
  3. Anokye-Danso et al., (2011) Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell, 8: 376-388. 
  4. Subramanyam D et al., (2011) Multiple targets of miR-302 and miR-373 promote reprogramming of humanfibroblasts to induced pluripotent stem cells. Nature Biotechnology., 29: 443-448.
  5. Miyoshi N et al., (2011) Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell., 8: 633-638.