- Reagents and protocols optimized to minimize endogenous RNase activity
- Superior identification of target cells provided by histological and fluorescent stains
- Designed for use with frozen, OCT®-embedded tissue sections (6-10 µm)
- Compatible with Arcturus LCM Systems
- Use with Ambion's RNAqueous®-Micro RNA Isolation Kit and MessageAmp™ II aRNA Amplification Kit for superior gene expression analysis
LCM for RNA Analyses
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
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)
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 visibility 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.
Quality RNA for Downstream Applications
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
Paul LeBourgeois, Ivonne Moon, Sharmili Moturi, Marianna Goldrick • Ambion, Inc.