Getting Started with microRNA Expression Analysis Research using Real-Time PCR
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:
- Effective method of miRNA isolation from samples
- RT-PCR reagents optimized for miRNA detection
- Assays specific to the miRNAs of interest
- 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)
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)
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)
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
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 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
A Complete Solution
Rick Conrad, Yvonne Potuceck, and Emily Zeringer • Ambion
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