miRNAs play critical roles in development, metabolism, and oncogenesis and are therefore tightly controlled both spatially and temporally. It is well established that miRNA levels vary from tissue to tissue [1-3], but little is currently known about cell type to cell type variation within a given tissue. Here, three Ambion kits were used to quantitate specific miRNAs and to detect differential miRNA expression in various mouse brain regions and cell types isolated by laser capture microdissection (LCM). These techniques can be applied to studying miRNA in other species, tissues, and cell types.
- LCM Staining Kit--optimized to avoid RNA degradation that commonly occurs during conventional LCM tissue processing steps [4]
- RNAqueous®-Micro Kit--designed for total RNA isolation from small samples, a simple procedural modification allows miRNA recovery
- mirVana™ qRT-PCR miRNA Detection Kit--sensitive detection and quantitation of miRNA by endpoint or real-time RT-PCR
The ability to analyze miRNAs in small populations of pure cells will significantly advance our understanding of the role of miRNAs in human health and disease. Starting with frozen, OCT®-embedded tissue samples, the following three steps quickly take you from sample preparation to miRNA quantitation:
1. Obtain Laser Capture Microdissected Samples. LCM is an ideal method that enables microdissection of specific cell populations from complex tissues. Traditional methods for tissue fixation and staining prior to LCM typically yield RNA of very poor quality. In contrast, the optimized protocol used by Ambion's LCM Staining Kit preserves RNA integrity by avoiding exposure of tissue sections to aqueous solutions, which promote reactivation of endogenous RNase activity. This kit includes two different stains (Cresyl Violet and Acridine Orange) that greatly facilitate target cell identification during LCM (Figures 1A-B and 2A-B) and lead to isolation of superior quality RNA compared to conventional protocols [4].
2. Isolate miRNA from LCM Samples. The RNAqueous-Micro Kit is ideal for purifying RNA from small samples, such as cell populations isolated via LCM [5]. Using glass-fiber filter purification technology, this kit has been optimized to isolate RNA from 10 cells to 10 mg of tissue. Adjusting the filter-binding conditions of the sample enables miRNA recovery in addition to larger RNA species (Figure 1C).
3. Quantitate miRNA by qRT-PCR. After total RNA is isolated from the LCM samples, the highly sensitive mirVana qRT-PCR miRNA Detection Kit can be used for quantitative detection of specific miRNAs. Along with a thermostable DNA polymerase (e.g., SuperTaq™ Polymerase) and the specially designed mirVana™ qRT-PCR Primer Sets, Ambion's miRNA detection assay permits miRNA amplification and detection by real-time RT-PCR using SYBR® Green I.
Figure 1. miR-124a Quantitation in Purkinje Cells Isolated Via LCM.(A) Mouse cerebellum sections (10 µm) were stained with Cresyl Violet using the LCM Staining Kit. LCM isolation of a single Purkinje cell is shown. (B) Three 10 µm sections of mouse cerebellum were used for the LCM. One representative section is shown here. About 200 Purkinje cells were selected from each section using the Arcturus PixCell®-II work station and pooled into a single RNAqueous®-Micro purification reaction, which yielded ~6 ng of total RNA. (C) Duplicate mirVana™ qRT-PCR Assays were performed using Purkinje cell total RNA (250 pg), a Primer Set specific for miR-124a, and SuperTaq™ Polymerase. In parallel, duplicate miR-124a assays were performed using a ten-fold dilution series (200 ng, 20 ng, 2 ng, and 200 pg) of FirstChoice® Mouse Brain Total RNA. 250 pg of Purkinje cell total RNA contained the same quantity of miR-124 as ~760 pg of FirstChoice Mouse Brain Total RNA, suggesting miR-124 expression is approximately three-fold higher in purified Purkinje cells than in whole mouse brain. (D) The dissociation curve shows a clear difference between primer-dimers generated in the No Template Control (NTC) and the single, desired amplicon derived from target miRNA.
1. Obtain Laser Capture Microdissected Samples. LCM is an ideal method that enables microdissection of specific cell populations from complex tissues. Traditional methods for tissue fixation and staining prior to LCM typically yield RNA of very poor quality. In contrast, the optimized protocol used by Ambion's LCM Staining Kit preserves RNA integrity by avoiding exposure of tissue sections to aqueous solutions, which promote reactivation of endogenous RNase activity. This kit includes two different stains (Cresyl Violet and Acridine Orange) that greatly facilitate target cell identification during LCM (Figures 1A-B and 2A-B) and lead to isolation of superior quality RNA compared to conventional protocols [4].
2. Isolate miRNA from LCM Samples. The RNAqueous-Micro Kit is ideal for purifying RNA from small samples, such as cell populations isolated via LCM [5]. Using glass-fiber filter purification technology, this kit has been optimized to isolate RNA from 10 cells to 10 mg of tissue. Adjusting the filter-binding conditions of the sample enables miRNA recovery in addition to larger RNA species (Figure 1C).
3. Quantitate miRNA by qRT-PCR. After total RNA is isolated from the LCM samples, the highly sensitive mirVana qRT-PCR miRNA Detection Kit can be used for quantitative detection of specific miRNAs. Along with a thermostable DNA polymerase (e.g., SuperTaq™ Polymerase) and the specially designed mirVana™ qRT-PCR Primer Sets, Ambion's miRNA detection assay permits miRNA amplification and detection by real-time RT-PCR using SYBR® Green I.
Figure 1. miR-124a Quantitation in Purkinje Cells Isolated Via LCM.(A) Mouse cerebellum sections (10 µm) were stained with Cresyl Violet using the LCM Staining Kit. LCM isolation of a single Purkinje cell is shown. (B) Three 10 µm sections of mouse cerebellum were used for the LCM. One representative section is shown here. About 200 Purkinje cells were selected from each section using the Arcturus PixCell®-II work station and pooled into a single RNAqueous®-Micro purification reaction, which yielded ~6 ng of total RNA. (C) Duplicate mirVana™ qRT-PCR Assays were performed using Purkinje cell total RNA (250 pg), a Primer Set specific for miR-124a, and SuperTaq™ Polymerase. In parallel, duplicate miR-124a assays were performed using a ten-fold dilution series (200 ng, 20 ng, 2 ng, and 200 pg) of FirstChoice® Mouse Brain Total RNA. 250 pg of Purkinje cell total RNA contained the same quantity of miR-124 as ~760 pg of FirstChoice Mouse Brain Total RNA, suggesting miR-124 expression is approximately three-fold higher in purified Purkinje cells than in whole mouse brain. (D) The dissociation curve shows a clear difference between primer-dimers generated in the No Template Control (NTC) and the single, desired amplicon derived from target miRNA.
Case Study:
Detection of miRNA in Microdissected Tissue from Mouse Brain by qRT-PCR
Using the techniques described above, Ambion kits were used to quantitate specific miRNAs in different regions of mouse brain. In brief, frozen mouse brain sections (10 µm) were processed with the LCM Staining Kit, and LCM samples were taken from various sub-regions including the forebrain (frontal cortex and caudate putamen), hippocampus (dentate gyrus, granular cells, and pyramidal cell layer), and cerebellum (Purkinje cells). After isolating total RNA containing miRNA with the RNAqueous-Micro Kit (alternative protocol for miRNA isolation), the mirVana qRT-PCR miRNA Detection Kit was used to quantitate various miRNAs.
miR-124a expression levels were characterized in Purkinje cells microdissected from the cerebellum of a mouse brain (Figure 1B). The mirVana qRT-PCR miRNA Detection Kit enabled miR-124a quantitation using total RNA isolated from a cluster of approximately 200 Purkinje cells using FirstChoice® Mouse Brain Total RNA as an external reference (Figure 1C). In addition, miR-99a expression levels were assayed in cells isolated from the dentate gyrus and pyramidal layer of the hippocampus (Figure 2).
Figure 2. miR-99a Quantitation in LCM Samples From the Dentate Gyrus and Pyramidal Layer of the Hippocampus. 10 µm sections from mouse midbrain were processed using the LCM Staining Kit and used for LCM of the dentate gyrus (A) and pyramidal layer (B) of the hippocampus using the Arcturus PixCell®-II work station. ~4 structures were selected per sample and isolated in a single RNAqueous®-Micro purification reaction. (C) qRT-PCR was performed as described for Figure 1C, except the Primer Set targeting miR-99a was used. In parallel, duplicate miR-99a assays were performed using a ten-fold dilution series (200 ng to 200 pg) of FirstChoice® Mouse Brain Total RNA. 250 pg of dentate gyrus RNA (left) and pyramidal layer RNA (middle) contained the same quantity of miR-99a as 670 pg and 320 pg of whole brain total RNA, respectively. The dissociation curve (right) shows a clear difference between the amplicons generated in the No Template Control (NTC) and the single amplicon derived from target miRNA.
Using the techniques described above, Ambion kits were used to quantitate specific miRNAs in different regions of mouse brain. In brief, frozen mouse brain sections (10 µm) were processed with the LCM Staining Kit, and LCM samples were taken from various sub-regions including the forebrain (frontal cortex and caudate putamen), hippocampus (dentate gyrus, granular cells, and pyramidal cell layer), and cerebellum (Purkinje cells). After isolating total RNA containing miRNA with the RNAqueous-Micro Kit (alternative protocol for miRNA isolation), the mirVana qRT-PCR miRNA Detection Kit was used to quantitate various miRNAs.
miR-124a expression levels were characterized in Purkinje cells microdissected from the cerebellum of a mouse brain (Figure 1B). The mirVana qRT-PCR miRNA Detection Kit enabled miR-124a quantitation using total RNA isolated from a cluster of approximately 200 Purkinje cells using FirstChoice® Mouse Brain Total RNA as an external reference (Figure 1C). In addition, miR-99a expression levels were assayed in cells isolated from the dentate gyrus and pyramidal layer of the hippocampus (Figure 2).
Figure 2. miR-99a Quantitation in LCM Samples From the Dentate Gyrus and Pyramidal Layer of the Hippocampus. 10 µm sections from mouse midbrain were processed using the LCM Staining Kit and used for LCM of the dentate gyrus (A) and pyramidal layer (B) of the hippocampus using the Arcturus PixCell®-II work station. ~4 structures were selected per sample and isolated in a single RNAqueous®-Micro purification reaction. (C) qRT-PCR was performed as described for Figure 1C, except the Primer Set targeting miR-99a was used. In parallel, duplicate miR-99a assays were performed using a ten-fold dilution series (200 ng to 200 pg) of FirstChoice® Mouse Brain Total RNA. 250 pg of dentate gyrus RNA (left) and pyramidal layer RNA (middle) contained the same quantity of miR-99a as 670 pg and 320 pg of whole brain total RNA, respectively. The dissociation curve (right) shows a clear difference between the amplicons generated in the No Template Control (NTC) and the single amplicon derived from target miRNA.
Case Study:
Differential Expression of MicroRNA in Whole Brain Tissue Compared to a More Homogeneous Population of Cells
To explore differential cell-specific miRNA expression levels, RNA samples were prepared from frontal cortex (from secondary visual cortex and retrosplenial granular areas) selected by LCM using a 10 µm thick coronal section of the midbrain area. Simultaneously, RNA was isolated from an adjacent intact coronal section on the same slide (both sections were processed with the LCM Staining Kit in parallel). Next, miRNA expression levels were compared within the above two samples to that of total RNA derived from whole mouse brain (FirstChoice Mouse Brain Total RNA).
Each RNA sample was analyzed in triplicate by mirVana qRT-PCR Assays using Primer Sets specific for three different miRNAs known to be expressed at high (miR-124a), medium (miR-99a), and low (miR-130b) abundance in brain (Figure 3). In addition, triplicate reactions were performed using the Primer Set specific for 5S rRNA, which provided an miRNA-independent reference for normalization. The relative expression levels of these three miRNAs were similar in the whole brain and intact coronal section, which confirmed that the LCM staining procedure did not skew miRNA recovery or otherwise adversely affect overall RNA quality. Interestingly, the frontal cortex cells isolated via LCM exhibited a distinct miRNA expression pattern. In particular, miR-99a was under-represented in the frontal cortex sample compared to the whole brain and intact coronal section.
Figure 3. The Frontal Cortex Exhibits Different miRNA Expression Patterns. Coronal sections (10 µm) of mouse brain were stained with Cresyl Violet from the LCM Staining Kit. RNA samples were prepared from cells picked from the frontal cortex or from an adjacent intact coronal section of the midbrain. Triplicate mirVana™ qRT-PCR Assays were performed using Primer Sets specific for 5S rRNA , miR-124a , miR-99a or miR-130b. For each RNA sample, the Ct value observed using the 5S Primer Set was subtracted from the Ct value for each miRNA-specific Primer Set (DCt). In this comparison of relative expression levels, a small DCt value denotes a higher miRNA expression level, whereas a large DCt value denotes lower miRNA expression. The pattern defined by these three miRNAs is similar in the FirstChoice® Mouse Brain Total RNA and intact coronal section, but is distinct in the frontal cortex.
To explore differential cell-specific miRNA expression levels, RNA samples were prepared from frontal cortex (from secondary visual cortex and retrosplenial granular areas) selected by LCM using a 10 µm thick coronal section of the midbrain area. Simultaneously, RNA was isolated from an adjacent intact coronal section on the same slide (both sections were processed with the LCM Staining Kit in parallel). Next, miRNA expression levels were compared within the above two samples to that of total RNA derived from whole mouse brain (FirstChoice Mouse Brain Total RNA).
Each RNA sample was analyzed in triplicate by mirVana qRT-PCR Assays using Primer Sets specific for three different miRNAs known to be expressed at high (miR-124a), medium (miR-99a), and low (miR-130b) abundance in brain (Figure 3). In addition, triplicate reactions were performed using the Primer Set specific for 5S rRNA, which provided an miRNA-independent reference for normalization. The relative expression levels of these three miRNAs were similar in the whole brain and intact coronal section, which confirmed that the LCM staining procedure did not skew miRNA recovery or otherwise adversely affect overall RNA quality. Interestingly, the frontal cortex cells isolated via LCM exhibited a distinct miRNA expression pattern. In particular, miR-99a was under-represented in the frontal cortex sample compared to the whole brain and intact coronal section.
Figure 3. The Frontal Cortex Exhibits Different miRNA Expression Patterns. Coronal sections (10 µm) of mouse brain were stained with Cresyl Violet from the LCM Staining Kit. RNA samples were prepared from cells picked from the frontal cortex or from an adjacent intact coronal section of the midbrain. Triplicate mirVana™ qRT-PCR Assays were performed using Primer Sets specific for 5S rRNA , miR-124a , miR-99a or miR-130b. For each RNA sample, the Ct value observed using the 5S Primer Set was subtracted from the Ct value for each miRNA-specific Primer Set (DCt). In this comparison of relative expression levels, a small DCt value denotes a higher miRNA expression level, whereas a large DCt value denotes lower miRNA expression. The pattern defined by these three miRNAs is similar in the FirstChoice® Mouse Brain Total RNA and intact coronal section, but is distinct in the frontal cortex.
Identify Cell- and Tissue-Specific miRNA Expression
These data underscore that even within a given tissue, distinct cell populations may exhibit different miRNA expression patterns. Therefore, powerful cell isolation and miRNA quantitation techniques (such as Ambion's LCM Staining Kit, RNAqueous-Micro Kit, and mirVana qRT-PCR miRNA Detection Kit) will be instrumental in future studies of the spatiotemporal regulation of miRNA expression.
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Scientific Contributors
Ivonne Moon, Eric Devroe, Emmanuel Labourier, Marianna Goldrick • Ambion, Inc