Identification of miRNA Profiles in Individual Embryonic Stem Cells
Characterizing the miRNA Profile in Embryonic Stem Cells
There is significant evidence that miRNAs are involved in the regulation of stem cell differentiation. Distinct sets of miRNAs have been found exclusively in ESCs or in differentiated adult tissues [1,2]. This research caught the attention of Drs. William Strauss (University of Colorado-Boulder) and Caifu Chen (Research and Development, Applied Biosystems Inc.) who collaborated to investigate whether miRNA expression profiles (signatures) unique to ESCs could be identified. These profiles could be used to characterize and distinguish totipotent ESCs from pluripotent cell types, such as embryoid body (EB) cells, and from terminally-differentiated somatic cells.
New Megaplex™ Primer Pools Enable miRNA Profiling From Single Cells
70 randomly selected individual cells from a mouse ESC line, day 3 EB cells, and several somatic cell types including 3T3 cells (a well-characterized transformed fibroblast cell line) and primary splenocytes were chosen for miRNA profiling. The cells were lysed by heating to 95°C for 5 minutes in 1X PBS. An early access version of Megaplex™ RT Primers—a pool of 237 stem-loop RT primers from the associated TaqMan MicroRNA Assays—was used to prime reverse transcription of lysates using the TaqMan MicroRNA RT Kit. Since the real-time PCR quantitation step required distributing this minute amount of starting material across 237 independent reactions, it was decided to incorporate a preamplification step. Preamplification preserves the distribution of miRNAs in the starting sample, in particular those miRNAs present at low copy number that would otherwise be diluted out when material was distributed to each independent real-time PCR assay, thereby maintaining overall assay sensitivity. The cDNA was preamplified using a limited number of PCR cycles and an early access version of Megaplex™ PreAmp Primers—a pool of PCR primers from the same 237 TaqMan MicroRNA Assays. Then the preamplification products were subjected to real-time PCR using the 237 individual TaqMan MicroRNA Assays. This workflow enabled single-cell analysis of the expression profiles of most of the murine miRNAs known at the time (miRBase v10, ). At the same time, expression data were collected for positive and negative controls, as well as for 21 mRNAs corresponding to transcripts recognized by the stem cell community for their association with ESC differentiation and self-renewal.
miRNA Levels Increase as Differentiation Proceeds
Figure 2. Single Cell Expression Profile of 237 miRNAs and 21 mRNAs in ES, EB, Splenocyte, and 3T3 Cells.
Megaplex Primer Pools and next generation TaqMan MicroRNA Arrays are now available from Applied Biosystems, making it possible for any laboratory to profile miRNA from minute amounts of starting materials. Samples such as tumor biopsies, fluorescently sorted cells, and laser capture microscopy specimens are now much more accessible, but these new tools offer the benefits of simple, rapid processing, and the sensitivity, specificity, and dynamic range of TaqMan real-time PCR to any research program that includes miRNA profiling.
Using Megaplex Primer Pools can substantially reduce sample handling; each set of Megaplex RT Primers contains up to 381 primers for reverse transcription of up to 381 miRNAs simultaneously in a single reaction. For very limited samples, such as those used in the research described here, or when maximum sensitivity is required, an optional preamplification step can be included using Megaplex PreAmp Primers. Although Megaplex Primer Pools can be run with plated individual TaqMan MicroRNA Assays, the ideal profiling workflow utilizes the companion TaqMan MicroRNA Array. The reverse transcribed, preamplified cDNA is simply combined with TaqMan Universal PCR Master Mix, loaded onto an array, and run on the Applied Biosystems 7900HT Fast Real-Time PCR System using universal cycling conditions.
In combination with TaqMan MicroRNA Arrays, Megaplex Primer Pools enable analysis of as little as 1 ng of input total RNA per reaction when run with a preamplification step. As described in this article, this technology can be pushed to significantly lower input amounts although currently RNA input amounts below 1 ng are not fully supported.
For both human and rodent species, comprehensive coverage of the Sanger miRBase v10 database content is achieved through a two TaqMan MicroRNA Array set, each associated with companion Megaplex Primer Pools.
Pairing TaqMan MicroRNA Arrays, Megaplex RT Primers, and PreAmp Primers provides a rapid method for miRNA profiling in human, mouse, or rat samples with minimal sample input requirements. Whether your profiling experiment requires ultimate sensitivity, broad coverage, or both; Megaplex Primer Pools offer the flexibility to reach your research goals.
2. Strauss W, Chen C, Lee C-T, Ridzon D (2006) Nonrestrictive developmental regulation of microRNA gene expression. Mamm Genome 17: 833–840.
3. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36: D154–D158.