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Stem Cell Focus microRNA PCR Panels

PCR panels containing miRCURY LNA™ Universal RT microRNA PCR assays for microRNAs related to stem cells. Panels are available in ready-to-use 96 and 384 well plates – just add cDNA and PCR master mix.

  • All relevant embryonic and induced pluripotent stem cell microRNAs in one PCR Panel
  • A highly flexible solution that allows you to focus on the microRNAs you need in your stem cell project
  • Profile all microRNAs using less than 20 ng total RNA
  • Sensitivity and specificity for accurate microRNA profiling
  • Fast and easy - ready-to-use format for direct sample application with a full profile in less than 3 hours
  • High compatibility – 96 and 384 well formats compatible with most real-time PCR instruments

microRNAs in stem cells

Stem cell research has received increasing attention in the recent years due to their notable potential to reproduce themselves as well as differentiate into many different cell types. Today, stem cell studies are applied within a wide range of research including understanding diseases, biomarker discovery and drug screening.

Nearly a decade ago, it was found that stem cell development is regulated by microRNAs. During the most recent years, it has become evident that microRNAs are key players in the regulation of stem cell differentiation and homeostasis. A better knowledge of how microRNAs regulate stem cell homeostasis and differentiation may be a key to getting a better understanding of development and a way to make increase the potential for stem cell based therapies.

Since embryonic and adult stem cells are significantly different with respect to microRNA expression patterns, Exiqon has designed a stem cell panel focusing on the pluripotent stem cells ensuring a proper coverage of all relevant microRNA for optimal use in embryonic stem cell research, e.g. for studying microRNAs which are differentially regulated and/or have functional relevance for stem cell differentiation and homeostasis as well as identification of biomarkers or molecular signatures for specific stem cells or stem cell differentiation processes. Exiqon’s Pluripotent Stem Cell Focus microRNA PCR Panel is based on microRNAs expressed in stem cells derived from human embryos (hESC) and induced pluripotent stem cells (iPSC). iPSCs are similar in phenotype to ESCs, but are developed by forced expression of transgenes in somatic cells. They were developed only recently and have received widespread attention since their development involves the use of reprogrammed somatic cells.

iPSCs and hESCs can be grown and maintained in vitro and differentiated into specialized cells when exposed to the right cocktail of stimuli. For adult stem cells, three major paths have been described: Mesoderm, Endoderm and Ectoderm. Mesoderm develops into skeleton muscles, bone and blood cells, whereas Endoderm develops into the different organs and finally Ectoderm develops into skin, tooth and nervous system (Figure 1).


Figure 1
Stem cell differentiation. (Click to learn more)



Product coverage

The miRCURY LNA™ Universal RT microRNA PCR, Pluripotent Stem Cell Focus microRNA PCR Panel is offered as a pre-defined Pick-&-Mix microRNA PCR Panel. It is a pre-defined list of assays for microRNAs expressed in human embryonic stem cells and induced pluripotent stem cells. Together, they comprise a panel that fits into a single 96-well PCR plate.



The panel contains:
  • 85 microRNA LNA™ PCR primer sets targeting human microRNAs expressed in human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC).
  • 3 of the microRNA assays can also be used as control primers: hsa-miR-103-3p, hsa-miR-191-5p and hsa-miR-423-5p.
  • 3 snRNA references genes (U6, SNORD38B, and SNORD49A). These 6 potential reference control genes are identical to those supplied with the Human Panel I+II, the Cancer Focus Panel, and the list for human (all assays) in the Pick-&-Mix Panel, making it easy to shift between panels.
  • 5 RNA spike-in controls available for selection in the Pick-&-Mix configurator for use as positive controls e.g. for quality control of the RNA purification and the RT reaction.
  • 3 inter-plate calibrators (3 x UniSp3 IPC).
Download a list of all microRNAs included on the panel (MS Excel)

A truly flexible solution

Since the panel is offered in the Pick-&-Mix configurator as a pre-defined panel, the panel composition is not static. Assays can be added or removed according to your needs. This highly flexible solution allows you to design the plates the way you want them in 96 or 384 well formats compatible with a wide range of PCR instruments (Figure 1).

How the assays were selected

All 85 stem cell specific microRNAs in the panel were selected based on experimental data from collaborative studies on stem cells with external partners using our miRCURY LNA™ Universal RT microRNA PCR system and results published in peer-reviewed journals. A total of 66 microRNAs are expressed in hESC, while 59 of the microRNAs are identified in iPSC. Hence, there is an overlap of 40 microRNAs which are both expressed in hESC and in iPSC (Figure 2). 



Figure 1
Instrument coverage of the panels. (Click to learn more)


Figure 2
Focus panel coverage. (Click to learn more)

Experimental data

See Figure 1 for a comparison of the microRNA expression of human embryonic stem cells and fully differentiated fibroblasts. The experiments were conducted using Exiqon's Pluripotent Stem Cell Focus microRNA Panels.

Figure 1
Comparison of microRNAs expression in stem cells and differentiated cells. (Click to learn more)



A unique system developed specifically for microRNA profiling

The miRCURY LNA™ Universal RT microRNA PCR System offers the best available combination of performance and ease-of-use on the market because it unites two important features (Figure 2):
  • One cDNA reaction for all microRNAs – One single Universal first strand cDNA synthesis reaction is used as template, regardless of the number of microRNAs being profiled. This saves precious sample, reduces technical variation, means less pipetting and saves time in the laboratory.
  • Two LNA™-enhanced microRNA qPCR primers – Both qPCR primers are microRNA-specific and optimized with LNA™. LNA™ primers bind with high affinity, and are shorter than standard PCR primers, so that both primers can fit on the microRNA without overlapping. The result is unrivalled sensitivity and specificity, and extremely low background.

Unmatched sensitivity

Universal RT combined with LNA™-enhanced and Tm normalized primers enables accurate and reliable quantification of individual microRNAs from as little as 1 pg total RNA (Figure 3). In comparison to other microRNA real-time PCR systems using either stem-loop or standard DNA primers, the LNA™-enhanced primers offer significantly increased sensitivity, especially for AU-rich microRNAs.

Exceptional sensitivity as well as extremely low background enables accurate quantification of very low levels of microRNA without the need for pre-amplification. This makes the miRCURY LNA™ Universal RT microRNA PCR System suitable for all sample types, and especially samples with low RNA content e.g. biofluids such as serum/plasma.

Fully validated and optimized

All miRCURY LNA™ Universal RT microRNA PCR primer sets have been optimized for maximum sensitivity and thoroughly validated either by wet lab testing or in silico. In wet lab validation, over 95% of assays detect 10 microRNA copies or less in the PCR reaction (Figure 4). The primer sets have also been validated for specific amplification of the target and for minimal background signal.

Truly specific – no false positives

The incorporation of LNA™ in the qPCR primers facilitates the design of assays that can distinguish between microRNA sequences that differ by a single nucleotide. This makes it a truly specific microRNA qPCR platform – the only microRNA platform that gives no false positive signals (Figure 5). In addition, the assays can discriminate between mature microRNA sequences and precursor microRNA (Table 1).

Fast, easy and reproducible

Save time and effort in the laboratory with the 3 hour easy-to-follow protocol that minimizes pipetting. By using the same cDNA as template in all subsequent PCR reactions, the procedure is greatly simplified compared to systems that require microRNA-specific cDNA synthesis.

With fewer pipetting steps, technical variation is also reduced. As a result, it is possible to achieve extremely high reproducibility from day-to-day (Figure 6) and even site-to-site.

Flexible qPCR system

Tailor your experimental setup to your specific need using individual assays or fully flexible custom Pick-&-Mix panels . Using the same cDNA reaction, shift seamlessly between individual primers, pre-designed miRNome and Focus panels, or custom Pick-&-Mix panels.
Figure 1 Overview of the miRCURY LNA™ Universal RT PCR workflow
Overview of the miRCURY LNA™ Universal RT PCR workflow. (Click to learn more)

Figure 2 Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System
Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System. (Click to learn more)

Figure 3 Accurate quantitation from down to 1 pg total RNA starting material
Accurate quantitation from down to 1 pg total RNA starting material. (Click to learn more)

Figure 4 Serial dilution of hsa-miR-181a
Serial dilution of hsa-miR-181a. (Click to learn more)

Figure 5 Specificity results from the miRQC Study
Specificity results from the miRQC Study. (Click to learn more)

Table 1 Discrimination between mature and precursor microRNAs
Discrimination between mature and precursor microRNAs. (Click to learn more)

Figure 6 Excellent day-to-day reproducibility
Excellent day-to-day reproducibility. (Click to learn more)

How to order Pluripotent Stem Cell Focus microRNA Panels

Pluripotent Stem Cell Focus microRNA Panels are ordered using our Pick-&-Mix microRNA PCR Panel plate configurator. Follow the steps below to order your plates.

1. Click on the button below. This will take you to our Pick-&-Mix microRNA PCR Panel plate configurator.


2. Register an account, sign in to your account or continue without signing in.

3. In Step 1 of the configurator, you are asked to choose between 96 and 384 well plates. Press “Next” to continue to the next step.

4. Select your PCR instrument and click “Next”.

5. In step 3 of the configurator, click the Stem Cell Focus Panel. You can select to include only hESC or iPSC microRNAs by clicking the “details” link and selecting the appropriate radio button. You will be able to alter your selection at a later stage. Press “Next”.

6. Please provide a name for your plate and enter your email address (email address is entered by default if you are signed in). Your email address is needed in order to send you the plate layout electronically. In this step you can also choose between different plate layouts.  For ordering the full Stem Cell  Focus Panel in a 96-well plate, you will need to select the 96 well – fully flexible layout . For ordering the full Stem Cell Focus Panel in a 384-well plate format you can choose between the “384-well – fully flexible” layout or the “94 microRNA x 4 samples” layout.  Press “Next”.
 
7. In step 5 of the configurator, you are asked to choose which assays to include on the plate. Press the “Assign all pre-defined assays to panel” button to include all assays. Please note, you will need to add the four additional RNA Spike-in controls manually (UniSp2, UniSp4, UniSp5 and cel-miR-39-3p). These are to be added in the four empty wells at the bottom of the panel. You can add or remove assays from the plate. To add an assay, click the check box next to the assay in the list to the left and press the “Upload to Panel” button. To remove individual assays, click on the well in the plate depiction to select it and then click “Delete Selected Assays”.

8. If you did not log in to your account before entering the plate configurator tool, you will be asked to do so now. In step 6 of the configurator, you can save your plate configuration, make corrections or order plates by pressing “Finalize plates”. This will place the plates in your shopping basket. From here, you can either buy the plates online or request a quote from your local sales representative or distributor if you are located in a country in which Exiqon has a distributor.



NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, nontransferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
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