Further development of Ambion's RNAqueous®-Micro Kit has resulted in a 3-6 fold increase in total RNA yields from LCM samples. In addition, Ambion has introduced a new LCM Staining Kit that has been optimized to avoid the RNA degradation issues commonly seen during conventional staining protocols. LCM tissues processed with Ambion's new LCM Staining Kit, followed by total RNA isolation using the improved RNAqueous-Micro Kit, generates high quality total RNA suitable for amplification with Ambion's MessageAmp™ II aRNA Amplification Kit. It is now possible to carry out microarray expression profiling and qRT-PCR using limited quantities of total RNA recovered from LCM samples. The experiments below illustrate the combined use of these Ambion products to enable expression profiling from LCM-microdissected brain samples.
Higher RNA Recovery from LCM Samples
The ability to analyze global expression profiles or specific gene expression levels from microdissected samples is becoming increasingly important for understanding biological processes. While the RNAqueous®-Micro Kit has always been ideal for isolating total RNA from microscale samples (<5 x 105 cultured cells and <10 mg tissue), Ambion scientists have now optimized its use for RNA recovery from microdissected samples. The improved kit shows a significant increase in total RNA yields isolated from microscale samples (e.g. 100 cells or LCM samples). The RNAqueous-Micro Kit generates high quality total RNA while keeping sample elution volumes low (10-20 µl). Radiolabeled, in vitro transcribed RNA was used to assess recovery of small amounts of RNA (0.01-10 ng) and variability introduced by differences in cell number in LCM samples. When this RNA was purified, the RNAqueous-Micro Kit showed significantly higher recovery of input RNA than a competitor's RNA isolation kit (Figure 1A). Improvements in RNA yield were also observed when isolating total RNA from ovarian tumor LCM samples using a combination of Ambion's LCM Staining Kit
and the RNAqueous-Micro Kit (Figure 1B).
Figure 1. The Improved RNAqueous®-Micro Kit Recovers More RNA. (A)
Radiolabeled GAPDH transcripts were generated with the MAXIscript® In Vitro Transcription Kit (Ambion). The mean percent recovery of RNA from dilutions containing 10-0.01 ng of radiolabeled GADPH transcripts were determined by scintillation counting after processing diluted RNA with two isolation kits. The mean percent recovery ± standard deviation for samples processed with the LCM Staining Kit (Ambion) and the RNAqueous®-Micro Kit (Ambion) was 80.4 ± 5.6 (n=8). (B)
RNA was isolated from ovarian tumor LCM samples (100 laser shots per sample) using Ambion's LCM Stains (Acridine Orange, Cresyl Violet) and RNAqueous-Micro Kit. The mean yields from the samples were determined by qRT-PCR using primers specific for human VEG-F and HPRT.
Microarray Analysis Using LCM Samples
RNA purified from LCM samples and processed with the RNAqueous-Micro Kit can be used for linear amplification and subsequent microarray analysis (Figure 2). Gene expression profiles comparing two mouse brain regions (hippocampus and cerebellum) were examined on an Affymetrix® GeneChip® array. The reproducible Percent Present Call rates (Figure 2A) indicated excellent, reproducible sensitivity when tissue sections were stained with either Acridine Orange or Cresyl Violet (LCM Staining Kit, Ambion). In addition, a scatter plot summarizing the overall results (Figure 2B) showed high correlation between expression results from both mouse hippocampus and cerebellar RNA. Using qRT-PCR, the results of the array experiments were validated for a small set of representative genes shown to be differentially expressed between hippocampus and cerebellum (Figure 2C).
Figure 2. Microarray Analysis of RNA from LCM Samples from Mouse Hippocampus and Cerebellum.
After staining with Cresyl Violet or Acridine Orange (LCM Staining Kit, Ambion), LCM samples were obtained from 10 µm sections of mouse brain with the PixCell® IIe LCM System (Arcturus). Two rounds of RNA amplification were performed with the MessageAmp™ II aRNA Kit (Ambion) using 20 ng of total RNA from each LCM sample. The resulting aRNA was labeled with biotin UTP and hybridized to an Affymetrix® Mouse 230A GeneChip® array. (A)
Graph summarizing Percent Present Call results for each sample. (B)
Scatter plot showing correlation between results from the two brain samples using the GCOS software (Affymetrix). (C) qRT-PCR using the TaqMan® Probe and Primer Set (Roche Molecular Systems) was used to validate the array data for nine genes with differential expression between hippocampus and cerebellum.
qRT-PCR from Small LCM Samples
RNA was isolated from mouse brain LCM samples from as little as five cells using the RNAqueous-Micro Kit (Figure 3A). A one-step qRT-PCR experiment was used to detected mFAS expression in the hippocampus (Ct = 32.6) (Figure 3B). It should be stated that the amount and integrity of total RNA isolated from any tissue sample is critically dependent on the type of tissue and the way in which it was harvested, stored, and treated prior to processing.
Figure 3. LCM Sample (~5 cells) from Mouse Hippocampus Can Be Analyzed by qRT-PCR. (A)
A 10 µm section of mouse hippocampus was stained with Acridine Orange (LCM Staining Kit, Ambion). Microdissection with the PixCell® IIe LCM System (Arcturus) was performed with the lowest settings (7.5 µm spot size, 40 mW power, 0.6 ms duration). Approximately five cells were captured. (40X magnification) (B)
RNA was isolated with the RNAqueous®-Micro Kit (Ambion) and the entire preparation (~32 pg) was used in a one-step qRT-PCR to amplify mFAS transcripts.
RNAqueous®-Micro Kit Components
The RNAqueous-Micro Kit provides reagents and supplies to process 50 LCM samples using a phenol-free, silica filter-based method. The kit also includes both reagents and protocols to remove trace amounts of genomic DNA that might interfere with downstream assays (e.g. RT-PCR), using an optional post-elution DNase treatment. Additionally, a simple modification in the protocol allows for the recovery of small RNA species including tRNA, 5S rRNA, and microRNAs.
Ivonne Moon, Sharmili Moturi, Marianna Goldrick • Ambion, Inc.