Protect RNA and Improve Target Cell Identification
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LCM for RNA Analyses
Laser capture microdissection (for overview see Laser Capture Microdissection (LCM): How its Done, at right) is a method that allows scientists to selectively collect pure cell populations from tissue sections. For RNA studies, frozen, OCT-embedded tissue is generally preferred over formalin-fixed, paraffin-embedded tissue--formalin fixation introduces cross-links between nucleic acids and proteins, resulting in RNA fragmentation. However, RNA extraction from frozen tissue is also complicated by reactivation of endogenous RNases that degrade RNA when tissue sections are exposed to aqueous solutions, as is the case with conventional staining protocols.
To overcome the technical problems of obtaining high-quality RNA from LCM samples, Ambion scientists have developed the LCM Staining Kit for frozen, OCT-embedded tissues. The LCM Staining Kit enables researchers to obtain high quality total RNA from small, homogenous samples particularly when used in combination with Ambion's RNAqueous-Micro RNA Isolation Kit, which now contains additional reagents and protocols for use with LCM samples.
To overcome the technical problems of obtaining high-quality RNA from LCM samples, Ambion scientists have developed the LCM Staining Kit for frozen, OCT-embedded tissues. The LCM Staining Kit enables researchers to obtain high quality total RNA from small, homogenous samples particularly when used in combination with Ambion's RNAqueous-Micro RNA Isolation Kit, which now contains additional reagents and protocols for use with LCM samples.
Superior Staining to Identify Cells of Interest
The LCM Staining Kit includes two stains that produce superior results compared to conventional protocols. These stains greatly facilitate target cell identification during LCM (Figure 1). Only Ambion's staining procedures avoid exposing tissue sections to aqueous solutions that reactivate endogenous RNase activity.
Figure 1. Obtain Better Resolution with Ambion's LCM Staining Kit Dyes. Consecutive sections from human small intestine (20X magnification) were stained according to manufacturers' instructions.
Figure 1. Obtain Better Resolution with Ambion's LCM Staining Kit Dyes. Consecutive sections from human small intestine (20X magnification) were stained according to manufacturers' instructions.
Cresyl Violet (Basic Nuclear Stain)
Cresyl Violet is a basic synthetic dye that binds acidic components such as RNA-rich ribosomes, nuclei, and nucleoli. This histological stain has been used to stain neurological tissues and is especially useful for allowing researchers to identify cellular morphology and tissue architecture (see Figure 2).
Figure 2. LCM Sample Stained with Cresyl Violet in Ambion's LCM Staining Kit. These representative photographs show an 8 µm section from human ovary (20X magnification) that was stained with Cresyl Violet and dissected with the Pixel® IIe LCM System (Arcturus). LCM sample Cluster of ovarian adenocarcinoma cells.
Figure 2. LCM Sample Stained with Cresyl Violet in Ambion's LCM Staining Kit. These representative photographs show an 8 µm section from human ovary (20X magnification) that was stained with Cresyl Violet and dissected with the Pixel® IIe LCM System (Arcturus). LCM sample Cluster of ovarian adenocarcinoma cells.
Acridine Orange (Fluorescent Intercalating Stain)
Acridine Orange stain intercalates into nucleic acids to enhance visability of nuclei in clusters of cells (Figure 3). Nuclear-rich epithelial tissues, such as clusters of malignant cells, are easily identified with this dye. Isolated cells stained with Acridine Orange will fluoresce on the cap after microdissection, making it easy to assess whether the cells were effectively microdissected and retrieved. If tissue morphology is slightly damaged, Acridine Orange may also provide better contrast than Cresyl Violet.
Figure 3. LCM Sample Stained with Acridine Orange in Ambion's LCM Staining Kit. These representative photographs show a 10 µm section from mouse hippocampus (4X magnification) that was stained with Acridine Orange and subsequently dissected with the Pixel® IIe LCM System (Arcturus). LCM sample Hippocampus granular cell layer and dentate gyrus.
Figure 3. LCM Sample Stained with Acridine Orange in Ambion's LCM Staining Kit. These representative photographs show a 10 µm section from mouse hippocampus (4X magnification) that was stained with Acridine Orange and subsequently dissected with the Pixel® IIe LCM System (Arcturus). LCM sample Hippocampus granular cell layer and dentate gyrus.
Quality RNA for Downstream Applications
The integrity of RNA isolated after staining samples with Ambion's unique dye formulation is superior to RNA isolated using a competitor's stain (Figure 4). RNA integrity was assessed on an Agilent® 2100 bioanalyzer. As seen in the electropherogram trace, a good 28S/18S rRNA ratio is maintained with human tumor samples stained with Cresyl Violet or Acridine Orange in the LCM Staining Kit (Figure 4). Size profiles also indicate less RNA degradation after staining samples with the LCM Staining Kit vs a competitor's staining kit. (Figure 4).
Figure 4. Higher RNA Quality with Ambion's LCM Stains. RNA was isolated from consecutive tissue sections that were either unstained or stained with three different dyes (Ambion's Cresyl Violet and Acridine Orange, and a Competitor Stain). The RNAqueous® Micro Kit (Ambion) was used to process samples that were stained with the LCM Staining Kit. The Competitor's RNA isolation kit and dyes were used to process separate samples. All of the resulting RNA preparations were analyzed on an Agilent® 2100 bioanalyzer. (A) Ratio of 28S rRNA to 18S rRNA of RNA preparations from various types of tumor samples. (B) Representative Agilent bioanalyzer traces for RNA preparations from human colon cancer sections show less degradation in the RNA isolated using the LCM Staining and RNAqueous-Micro Kits (top) compared to RNA isolated with the competitor's reagents (bottom).
RNA from LCM samples stained with Cresyl Violet or Acridine Orange can be used to analyze gene expression with either qRT-PCR or array analyses (Figure 5). The RNAqueous-Micro Kit was used to isolate RNA from the LCM sample shown in Figure 2. RNA from HPRT was amplified from this sample by qRT-PCR (Figure 5A). In addition, the RNAqueous-Micro Kit was used to isolate RNA from the LCM sample shown in Figure 3. This RNA (20 ng) was linearly amplified using the MessageAmp™ II aRNA Amplification Kit (Cat# 1751) and used for subsequent microarray analysis. Agilent bioanalyzer results show that yield (mean ± SD, 142 ± 12 µg) and size distribution (median, 1100 nt) of the aRNA were excellent in this experiment (Figure 5B).
Figure 5. Representative Results from qRT-PCR and aRNA Amplification Experiments from LCM Samples. (A) One-step qRT-PCR reaction to amplify HPRT mRNA was performed with total RNA isolated from human ovary LCM sample after staining with Cresyl Violet (see Figure 2; LCM Staining Kit, Ambion). (B) The MessageAmp™ II aRNA Kit (Ambion) was used to amplify 20 ng of total RNA isolated from mouse hippocampus and cerebellum after staining with Acridine Orange (AO) (see Figure 3) and Cresyl Violet (CV) (LCM Staining Kit, Ambion).
Figure 4. Higher RNA Quality with Ambion's LCM Stains. RNA was isolated from consecutive tissue sections that were either unstained or stained with three different dyes (Ambion's Cresyl Violet and Acridine Orange, and a Competitor Stain). The RNAqueous® Micro Kit (Ambion) was used to process samples that were stained with the LCM Staining Kit. The Competitor's RNA isolation kit and dyes were used to process separate samples. All of the resulting RNA preparations were analyzed on an Agilent® 2100 bioanalyzer. (A) Ratio of 28S rRNA to 18S rRNA of RNA preparations from various types of tumor samples. (B) Representative Agilent bioanalyzer traces for RNA preparations from human colon cancer sections show less degradation in the RNA isolated using the LCM Staining and RNAqueous-Micro Kits (top) compared to RNA isolated with the competitor's reagents (bottom).
RNA from LCM samples stained with Cresyl Violet or Acridine Orange can be used to analyze gene expression with either qRT-PCR or array analyses (Figure 5). The RNAqueous-Micro Kit was used to isolate RNA from the LCM sample shown in Figure 2. RNA from HPRT was amplified from this sample by qRT-PCR (Figure 5A). In addition, the RNAqueous-Micro Kit was used to isolate RNA from the LCM sample shown in Figure 3. This RNA (20 ng) was linearly amplified using the MessageAmp™ II aRNA Amplification Kit (Cat# 1751) and used for subsequent microarray analysis. Agilent bioanalyzer results show that yield (mean ± SD, 142 ± 12 µg) and size distribution (median, 1100 nt) of the aRNA were excellent in this experiment (Figure 5B).
Figure 5. Representative Results from qRT-PCR and aRNA Amplification Experiments from LCM Samples. (A) One-step qRT-PCR reaction to amplify HPRT mRNA was performed with total RNA isolated from human ovary LCM sample after staining with Cresyl Violet (see Figure 2; LCM Staining Kit, Ambion). (B) The MessageAmp™ II aRNA Kit (Ambion) was used to amplify 20 ng of total RNA isolated from mouse hippocampus and cerebellum after staining with Acridine Orange (AO) (see Figure 3) and Cresyl Violet (CV) (LCM Staining Kit, Ambion).
LCM Staining Kit Components
Each kit contains reagents sufficient for processing 80 slides with either stain. Additional components have been included in the kit that improve both staining quality and microdissection. For example, the kit comes with a barrier pen that is used to position the stains on the tissue sections; dehydration beads are provided to facilitate dehydration of the tissue--critical for successful LCM using the Arcturus instrument; RNase-free vessels hold the slides during processing; and RNaseZap® and Nuclease-free Water minimize tissue sample exposure to environmental RNases. A CD that demonstrates several crucial steps in the tissue sectioning, processing, and staining protocols is also included. Users only need to supply ACS-grade ethanol, xylenes, and slides with samples.
Scientific Contributors
Paul LeBourgeois, Ivonne Moon, Sharmili Moturi, Marianna Goldrick • Ambion, Inc.
Scientific Contributors
Paul LeBourgeois, Ivonne Moon, Sharmili Moturi, Marianna Goldrick • Ambion, Inc.
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