Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
|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
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
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
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
Ivonne Moon, Sharmili Moturi, Marianna Goldrick • Ambion, Inc.