Getting Started with microRNA Expression Analysis Research using Real-Time PCR (Ambion TechNotes 13:4)

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TechNotes 13(4)

mirVana™ miRNA Isolation Kit (Ambion)
TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems)
TaqMan® MicroRNA Assays (Applied Biosystems)
Getting Started with microRNA Expression Analysis Research using Real-Time PCR

Often one of the first questions addressed when starting microRNA (miRNA) research is “Is there a specific subset of miRNAs expressed across my system?” e.g., in tumor vs. normal adjacent tissue, or across different stages of development. Here, we provide a general overview for identifying differentially expressed miRNAs. We also describe some of the specialized research tools that can help assess miRNA expression.

Introduction
microRNAs (miRNAs) are small, highly conserved RNA molecules that act as key regulators of development, cell proliferation, differentiation, and the cell cycle. The active, mature miRNAs are 17–24 base, single-stranded RNA molecules expressed in eukaryotic cells that are known to affect the translation or stability of target messenger RNAs. Each miRNA is believed to regulate multiple genes--greater than one third of all human genes may be regulated by miRNA molecules [1].

In order to gain insight into these tiny regulators, researchers around the world are asking fundamental research questions such as: "What miRNAs are expressed?”, “Where and when are they expressed?”, "What cell processes do miRNAs regulate?" and "What genes do miRNAs control?”.

Now that many miRNA sequences are known (catalogued in the miRBase Sequence Database), one of the most common next steps is analysis of miRNA expression levels between different tissues, developmental stages, or disease states. miRNA expression levels can be studied by several methods: microarray analysis, real-time PCR, Northern blots, in situ hybridization, and solution hybridization. Of these techniques, quantitative reverse transcription PCR (qRT-PCR) is the most sensitive and accurate method.

miRNAs can be a challenge to study because of their small size. They require specialized and dedicated tools for analysis. For qRT-PCR applications the tools include:

1. Effective method of miRNA isolation from samples

2. RT-PCR reagents optimized for miRNA detection

3. Assays specific to the miRNAs of interest

4. Real-time analytical instruments and reagents validated for miRNA detection protocols

Here we provide an experimental overview for quantitating specific miRNA expression levels by qRT-PCR (outlined in Figure 1). In this study, we analyzed miRNA levels from total RNA and RNA samples enriched for small RNA. These samples included both frozen or RNAlater® Solution-treated mouse, brain, liver, and lung tissues.

Figure 1. miRNA Experimental Overview.

1. Effective method of miRNA isolation from samples (mirVana™ miRNA Isolation Kit)

A. Sample Acquisition and Storage
Once samples have been obtained, they should be processed immediately--tissue should be frozen (small pieces in liquid nitrogen is preferable), or placed in RNAlater® Solution for storage until RNA extraction is performed. RNAlater Solution is an aqueous tissue storage reagent that protects RNA within intact, fresh samples.

Frozen mouse brain (Cat #55004-2), liver (Cat #55023-2), and lung (Cat #55024-2) tissues were acquired from Pel-Freeze Biologicals. Fresh brain, liver, and lung tissues stored in RNAlater Solution were extracted and processed from C57BL/6J male mice from The Jackson Laboratory (Cat #1628517).

B. Isolation of Total RNA with microRNA Using the mirVana miRNA Isolation Kit
Isolation of miRNA begins when total RNA that includes the small RNA fraction is isolated from the samples of interest. However, not all isolation methods retain the small RNA fraction. Therefore it is important to use isolation methods specifically adapted for retaining small RNA species. The mirVana™ miRNA Isolation Kit was developed to retain small RNA species either in a background of total RNA or as an enriched fraction of RNA species, 200 nucleotides or smaller. The initial organic extraction of the mirVana miRNA Isolation Kit provides a robust front-end purification that removes cell debris and most DNA. Although enrichment of the small RNA fraction can increase sensitivity in many applications, total RNA is usually sufficient for qRT-PCR detection of miRNA.

In our experiment, total RNA was isolated from the brain, liver, and lung tissue of three mouse specimens (Figure 2). The tissue samples were disrupted in lysis buffer and then acid-phenol:chloroform extracted, following the mirVana miRNA Isolation Kit procedure. Final extract volumes were measured in preparation for the second phase of the procedure. Total RNA was then purified by adding ethanol to the samples and passing them through a glass-fiber filter (GFF), which immobilized the RNA. The filter was washed a few times and the total RNA eluted using a low ionic-strength solution.

Figure 2. Efficient Recovery of miRNA. Total RNA was isolated from brain, liver, and lung tissue using the mirVana™ miRNA Isolation Kit. Typically, one can expect to get about 1 µg RNA for every milligram of tissue. The mirVana Isolation Kit also provides reagents and a procedure to enrich the population of RNAs that are 200 bases and smaller. Since tRNA and other small functional RNAs comprise 5–20% of the total RNA population, the gross recovery of enriched RNA by A260 will only be about a tenth of that total, but the amount of miRNA present will be the same.

Figure 2 demonstrates efficient recovery of total RNA and enriched miRNA from tissues of 3 animals using the mirVana Isolation Kit. About 1 µg of total RNA was recovered in each animal for each tissue type. Because tRNA and other small functional RNAs comprise 5−20% of the total RNA population, the gross recovery of enriched small RNA by A260 was about 1/10th that of the total RNA isolated, but the amount of miRNA present in the total RNA versus the enriched fraction was about the same.

C. Yields and Quality of RNA
Typically, yields for total RNA follow the “1/1000th rule”, i.e., one can expect to get about 1 µg of RNA for every milligram of tissue. This rule varies with tissue type, e.g., skin yield is much less, but most yields are within a 5-fold level. The mirVana Isolation Kit provides reagents and a procedure to enrich the population of RNAs that are 200 bases and smaller, using two sequential filtrations through GFFs with different ethanol concentrations. Although generally not necessary for real-time PCR applications, small RNA enrichment results in lower background and enhanced sensitivity of small RNA detection by solution hybridization, Northern analysis, and other methods, compared to the same assay using total RNA.

RNA yield, purity, and quality are factors that are important for successful gene expression analysis. RNA yield can be measured by looking at the A260 reading. A reliable and inexpensive method for determining RNA quality is to run the samples on a polyacrylamide gel. In this experiment, 250 ng RNA from 1 biological replicate set was combined with 5 µL of Ambion’s Gel Loading Buffer II (Ambion Cat #AM8546G) and concentrated using a Savant SpeedVac® on medium heat to a final volume of 10 µL. Samples were then incubated for 2 minutes at 95°C and immediately placed on ice (Figure 3). Decade™ Marker was prepared according to protocol using Ambion’s mirVana™ Probe & Marker Kit (Ambion Cat #AM1554). Samples were run on a polyacrylamide gel.

Figure 3. RNA Yield in Frozen Versus RNAlater® Solution-Treated Samples. The greatly enriched presence of tRNA (~70 nt) is apparent, as equal amounts of RNA were loaded. The RNAlater® solution-treated samples (A) provide equivalent samples in terms of banding patterns when compared to frozen samples (B). The enrichment procedure is not totally size-dependent, but also enriches for some small RNAs preferentially (perhaps due to structural qualities). The mass of large RNAs (trapped in the gel origin) are greatly reduced. The lower molecular weight bands seen in the lung samples are occasionally seen in this sample type, and could be degradation products. Each sample was run on a 7 M urea/15% polyacrylamide gel with 1 µL unlabeled Decade™ Markers (MKRS; Ambion; Cat #AM7778). Prior to sample loading, gels were run at 300 V for 10 min, and the wells were flushed with 1X TBE Buffer. The gel was run at 200 V until exit of the bromophenol blue dye front from the gel. Gels were stained for 30 minutes with a 1:10,000 dilution of SYBR® Gold Dye and photographed using Alphaimager v5.5 software. Sample loading was standardized according to ng RNA loaded.

Figure 3 illustrates the yield of the varying RNA sizes run on a polyacrylamide gel, in which losses in yield were caused by losing the high molecular weight RNA species that were embedded in the gel at the origin. Here also the enriched RNA was about 1/10th that of the total RNA.

2. Optimized RT-PCR Reagents for miRNA Detection (TaqMan® MicroRNA Reverse Transcription Kit)
Gene expression studies require high quality cDNA. The Applied Biosystems TaqMan® MicroRNA Reverse Transcription Kit delivers accuracy, precision, and quality. The kit provides the necessary components for optimal performance of TaqMan MicroRNA Assays. Components of this kit are used with the RT primers in the TaqMan MicroRNA Assays to convert specific miRNAs to cDNA.

In this experiment, an RNA mass equivalent of 5.13 µg of tissue was added to a final RT reaction volume of 15 µL. RT was performed in 384 well format using the TaqMan MicroRNA RT Kit protocol.

3. Assays Specific to the miRNAs of Interest (TaqMan® MicroRNA Assays)
Until recently, small RNAs like miRNA should not be analyzed by traditional PCR. Applied Biosystems TaqMan® MicroRNA Assays have been validated to amplify specific small RNAs, enabling accurate and specific quantitation of miRNA expression levels. A large collection of pre-designed, off-the-shelf TaqMan MicroRNA Assays for human, mouse, rat, A. thaliania, C. elegans, and D. melanogaster are available from Applied Biosystems. Each one is functionally validated, convenient, and easy to use.

In our study, the following TaqMan MicroRNA Assays and controls were used: hsa-miR-24, hsa-miR-16, hsa-miR-145, RNU6B (U6 Control), and RNU19 (U19 Control). The miRNAs to which these assays were designed have been shown to exhibit differential expression patterns in cancerous tissues as compared to normal tissues and may play a role in oncogenesis [2−4].

4. Real-time analytical instruments and reagents validated for miRNA detection protocols (Applied Biosystems 7900HT Fast Real-time PCR System, and TaqMan® Universal PCR Master Mix)
Applied Biosystems Real-Time PCR Systems make real-time PCR more accessible than ever before by providing powerful solutions to fit the needs of any laboratory. By taking advantage of gold-standard TaqMan® reagent-based technology with universal thermal cycling conditions, TaqMan Universal PCR Master Mix is the ideal reagent solution for TaqMan assays using DNA or cDNA as the target. The TaqMan Universal PCR Master Mix and TaqMan Reagent technology have been validated for use with TaqMan MicroRNA Assays.

In our experiment, real-time PCR was performed by adding 1.34 µL (a 458 ng tissue equivalent) of each completed RT reaction to a target TaqMan MicroRNA Assay reaction using TaqMan Universal PCR Master Mix (final reaction volume equal 20 µL) (Figure 4). Samples were tested in triplicate and run on the Applied Biosystems 7900HT Fast Real-Time PCR System. Assay results were collected and analyzed using SDS 2.2.2 software.

Figure 4. Real-time PCR Results in Frozen Versus RNAlater® Solution-Treated Sample. The frozen and RNAlater solution-treated samples are roughly equivalent, and the enriched samples show ~3.3 Ct’s increase in signal, consistent with about a tenfold enrichment. U6 and U19 are TaqMan® MicroRNA Assay Controls, which have been designed to aid in relative quantitation. *hsa-miR-133a (Panel A) and hsa-miR-1 (Panel B) were spiked into the samples as controls.

Results and Conclusions
Figure 4 shows a ~3.3 Ct difference between miRNA levels in the total RNA samples and the samples enriched for miRNA, which indicates about a tenfold enrichment. This is consistent with our expectations given that the data were normalized to input mass of RNA. Of note, the differential miRNA expression level trends between the different tissues were similar in both sample types. This experiment, as well as others done in our labs, demonstrates that miRNA enrichment preserves miRNA differential expression patterns as compared to total RNA. As mentionted earlier, although we find that total RNA isolated with the mirVana miRNA Isolation Kit yields more than sufficient signal with TaqMan MicroRNA Assays, other techniques such as microarray and Northern analysis require this enrichment to yield sufficient signal for analysis.

Figure 4 also indicates that the frozen and RNAlater® solution treated samples yielded Cts that were roughly equivalent. This experiment demonstrates that there is no significant difference in miRNA expression profiles from frozen and RNAlater solution-treated tissues when RNA is isolated using the mirVana miRNA Isolation Kit.

The Next Step: Functional Analysis
To date, the functional roles of only a handful of miRNAs have been determined. These roles have been shown to encompass a wide range of biological functions ranging from developmental timing (e.g., lin-4 in C. elegans), to apoptosis (e.g., bantam in Drosophila), to tissue growth (e.g., JAW in plants) [2,3]. These new findings suggest that the functions of miRNAs also include a role in asymmetric gene expression [5]. Specific miRNA function can be examined by up and down regulating specific miRNA levels to study endogenous or reporter gene regulation and phenotypic response. miRNA functional analysis can be performed by using synthetic miRNA mimics (Pre-miR™ miRNA Precursors; Ambion) and miRNA inhibitors (Anti-miR™ miRNA Inhibitors; Ambion) which are now available for most known miRNAs.

A Complete Solution
Applied Biosystems (including Ambion, The RNA Company®) offers a complete suite of reagents, instruments and protocols dedicated to the investigation of miRNAs. In this study we have shown that together, the mirVana miRNA Isolation Kit, TaqMan® MicroRNA RT Kit, TaqMan MicroRNA Assays, and 7900HT Fast Real-Time PCR System provide a validated, reliable and ready-to-use approach for quantitation of microRNA levels from a variety of sample types. We envision that this validated miRNA expression profiling platform will accelerate discovery of the many biological roles of miRNAs in cells, and more importantly, their roles in human diseases.

For more information about the entire suite of microRNA analysis tools from Applied Biosystems, visit the miRNA Resource.

Figure 5. miRNA Fundamental Research Questions.

Scientific Contributors
Rick Conrad, Yvonne Potuceck, and Emily Zeringer • Ambion

Ordering Information for Ambion Products:

Cat#	Product Name	Size
AM1554	mirVana™ Probe & Marker Kit	30 rxns + 10 marker rxns
AM1560	mirVana™ miRNA Isolation Kit	up to 40 purifications
AM17000	Anti-miR™ miRNA Inhibitor	5 nmol
AM17100	Pre-miR™ miRNA Precursor Molecule	5 nmol
AM7020	RNAlater®	100 ml
AM8546G	Gel Loading Buffer II (Denaturing PAGE)	1.4 ml
For Research Use Only. Not for use in diagnostic procedures.

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References

1. Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1):15–20.

2. Esquela-Kerscher A, Slack FJ (2006) Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 6(4):259−269.

3. Hammond SM (2006) MicroRNAs as oncogenes. Curr Opin Genet Dev 16(1):4−9.

4. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M et al (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 103(7):2257−2261.

5. Johnston RJ, Hobert O (2003) A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans. Nature 426:845−849.

How Does a Real-time PCR Assay Work?
A real-time PCR assay is a tool that can be used to quantitate differential gene expression. This type of assay measures (quantitates) the amount of a nucleic acid target during each amplification cycle of the PCR. The target may be DNA, cDNA, or RNA. In the initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline for the amplification plot. An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. A fixed fluorescence threshold can be set above the baseline. The parameter Ct (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. Relative quantitation might be used to measure gene expression in response to a chemical (drug). The level of gene expression of a particular gene of interest in a chemically treated sample would be compared relative to the level of gene expression in an untreated sample. Another method of looking at gene expression using quantitative reverse transcription PCR is to look at absolute quantitation and look at standard curves made using a dilution curve.

Fundamental Research Questions
As a unique class of small RNA molecules, miRNAs require special tools for accurate and sensitive analysis. Applied Biosystems scientists (now including Ambion, The RNA Company®) have developed a portfolio of products that provide a complete solution to accelerate identification and characterization of this class of tiny RNA regulators (Figure 1). These tools will help scientists answer fundamental miRNA research questions such as: which miRNAs are expressed, where and when are they expressed, what cell processes do miRNAs regulate, and which genes do miRNAs control (Figure 5). The relationship between mRNA and protein expression can now be easily studied, and this may lead to a new segment of targets for diagnostics and therapeutics.