Navigation Links
Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis

RNAqueous-Micro Kit Ideal for Processing Laser Capture Microdissection Samples


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 l). 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). The article Protect RNA and Improve Target Cell Identification provides further data illustrating the high quality of total RNA isolated from LCM samples with this kit.

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).


[click to enlarge]

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 m 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 UT P 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 m 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 m 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.


Scientific Contributors
Ivonne Moon, Sharmili Moturi, Marianna Goldrick Ambion, Inc.


back to top


Ordering Information
Cat# Product Name Size 1931 RNAqueous-Micro Kit 50 purifications 1935 LCM Staining Kit Reagents for processing 80 slides
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
2. Restriction Digests of DNA Isolated with the Perfectprep Gel Cleanup Kit
3. A Better Way to Isolate RNA AND Protein From the Same Sample
4. Isolate It All: siRNA miRNA Total RNA Native Protein
5. Isolate Total RNA and Protein From the Same Sample
6. A Simple and Rapid Technique to Purify High-Quality Midiprep DNA
7. Generate High-Quality, Directional Plasmid cDNA Libraries
8. Quality control of antibodies using the 2100 bioanalyzer and the Protein 200 Plus assay
9. Quality assurance of RNA derived from laser microdissected tissue samples obtained by the PALM MicroBeam System using the RNA 6000 Pico LabChip kit.
10. High Quality RNA Isolation Services
11. Preparation of PCR Quality Template DNA from a Wide Range of Tissues Using InstaGene Matrix
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:12/9/2016)... ... 09, 2016 , ... Aditya Humad, Acting CFO of AxioMed and Managing Partner ... United States, Axiomed is now gaining interest from Silicon Valley. “It was satisfying to ... went on to say that, “We expect interest to continue to rise as AxioMed ...
(Date:12/9/2016)...  A platform of orthopedic device technologies developed by ... closer to becoming viable options for repairing broken ... start-up company from Philadelphia, PA. ... novel absorbable bone fixation and regenerative repair technologies ... The company has leased space within the University ...
(Date:12/9/2016)... - Portage Biotech Inc. ("Portage" or "the Company") (OTC: ... that Biohaven has issued today the following press release: ... (PRWEB) Dec 9, 2016 - Biohaven Pharmaceutical Holding Company ... U.S. Food and Drug Administration ("FDA") has granted the ... BHV-0223, an orally dissolving tablet being developed for the ...
(Date:12/8/2016)... Board of Directors of the Pittsburgh Life Sciences ... only pure life sciences investment firm, today announced a ... developed by the Nominating and Governance Committee over the ... Jordan is selected to serve as President and ... who is elected to the position of Executive Chairman ...
Breaking Biology Technology:
(Date:11/17/2016)... 2016  AIC announces that it has just released a new white paper authored ... scale-out plus high speed data transfer storage solutions. Photo - http://photos.prnewswire.com/prnh/20161116/440463 ... ... ... Setting up a high performance computing or HPC system ...
(Date:11/15/2016)... , Nov 15, 2016 Research and Markets ... Forecast to 2021" report to their offering. ... ... 16.18 Billion by 2021 from USD 6.21 Billion in 2016, growing ... Growth of the bioinformatics market is driven by the growing ...
(Date:6/27/2016)... , June 27, 2016 Research and ... North America 2016-2020" report to their offering. ... North America to grow at a CAGR of ... been prepared based on an in-depth market analysis with inputs from ... prospects over the coming years. The report also includes a discussion ...
Breaking Biology News(10 mins):