Navigation Links
Analysis of Microarray Data

Data Generated From Both mRNA
and aRNA Samples

Clustering analysis is commonly used for interpreting microarray data. It provides both a visual representation of complex data and a method for measuring similarity between experiments (gene ratios). The widely used methods for clustering microarray data are: Hierarchical, K-means and Self-organizing map. In this article, the second in our series on Ambion's MessageAmp aRNA Amplification Kit, we present data and statistical analyses from experiments conducted by Drs. Philip Moos and Brian Dalley at the University of Utah, Huntsman Cancer Institute (HCI). The microarrays used in this study were manufactured at the HCI and contained 6912 cDNA clones deposited in duplicate using a Molecular Dynamics GEN III Array Spotter. Moos and Dalley compared the data generated from the HCI microarrays hybridized with mRNA and amplified antisense RNA (aRNA) generated with the MessageAmp Kit.

Experimental Design
Total RNA was isolated from HCT116 and RKO cells and aRNA was amplified from total RNA (2 g). For non-amplified samples, mRNA was purified using Oligotex beads (Qiagen). To assess the reproducibility of RNA amplification and compare representation of messages in aRNA with those in mRNA, three samples each of RKO and HCT116 RNA were amplified independently. The quality and yields of RNA obtained from the various samples are presented in Figure 2. mRNA or aRNA (2 g) samples were fluorescently labeled by incorporating Cy3-dCTP (RKO samples) or Cy5-dCTP (HCT 116 samples) during reverse transcription with random 9-mers. Each glass slide contained duplicate arrays and each labeled RNA sample was hybridized to two slides (4 replicates). Duplicate hybridizations were performed with each sample RNA and the quantified data was represented as slide averages resulting in 12 arrays for each aRNA and mRNA sample. Images were acquired using a Molecular Dynamics Array Scanner. Microarray data analysis was performed using Spotfire Software (Spotfire, Somerville, MA).

Cluster Analysis
An example of the image data obtained from 4 of the 12 grids from a standard 6912 element HCI array is represented in Figure 1. Comparison of the signals obtained using mRNA vs. aRNA indicates that RNA amplification provides excellent signal to noise, even for genes with a low level of expression.

Figure 1. Array Analysis of 6912 Element Arrays. Image Data from 4 of the 12 grids of a standard 6912 element Hunstman Cancer Institute cDNA microarray. mRNA or aRNA (2 g) was reverse transcribed in the presence of random ninemers and Cy dyes. HCT 116 samples were labeled with Cy5-dCTP while RKO cells were labeled with Cy3-dCTP. Analysis of the microarray data was carried out using Spotfire Software (Spotfire, Somerville, MA).

mRNA Sample A260/280 aRNA Yield RKO-1 2.12 37.0 g RKO-2 2.15 28.7 g RKO-2 2.12 26.8 g HCT116-1 2.12 33.5 g HCT116-2 2.12 36.2 g HCT116-3 2.12 29.5 g

Figure 2. Yields of Amplified RNA. Yields obtained after amplification of total RNA (2 g) from HCT116 and RKO cells using the MessageAmp Kit.

Hierarchical clustering analysis of the data obtained from 6912 elements was carried out using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis (see sidebar "Clustering Methods Used for Analyzing Microarray Data"), with an ordering function based on the input rank. This data is represented as a dendrogram (tree graph) with the closest branches of the tree representing arrays with similar gene expression patterns. Figure 3 depicts the hierarchical clustering data from all 6912 elements. The results indicate that there are broad similarities between arrays hybridized with aRNA or mRNA. Even though the overall signal patterns found on the aRNA and mRNA hybridized arrays are similar, a small subset of regions show differential expression (RKO/HCT116) signals between the aRNA and mRNA samples.

Figure 3. Hierarchical Clustering Analysis of All Array Elements. Hierarchical clustering data of all the elements in a HCl array. A dendrogram (tree graph) epicts the grouping of the genes based on the similarity between them. UPGMA analysis (unweighted average) was carried out using the "Euclidean Distance" to determine the similarity measure and the input rank as the ordering function. A subset of all the columns constituting the complete data is shown in this figure.

To obtain statistically significant data for the sub-regions that were distinct between the aRNA and mRNA (91 elements), a weighted average (WPGMA) analysis was carried out. The hierarchical clustering of these 91 elements is depicted in Figure 4. It is evident that there are very few genes that clearly segregate into either mRNA or aRNA groups. It is important to note, for those genes that do segregate, the gene expression differences (ratios) do not change direction (i.e. RKO>HCT to HCT>RKO), but show greater differences in the aRNA samples compared to the mRNA samples (as determined by the color shade).

Figure 4. Hierarchical Clustering Analysis of Selected Array Elements. Hierarchical clustering analysis of a few select genes (91 of 6912) that are very different between the aRNA and mRNA samples. This analysis was carried out using the weighted average (WPGMA) method.

An alternate methodology used to understand the clustering of microarray data is k-means clustering. This method does not suffer from some of the problems associated with hierarchical clustering such as irrelevance of gene expression data as clustering progresses or spurious results due to errors in assigning clusters initially in the analysis (2). K-means clustering of all the elements of the HCI arrays with 6 clusters was determined (Figure 5). After 45 iterations, a total score of 1.082e+004 was computed. The most similar "similarity value" was 0 and the least similar "similarity value" was 1.798e+308. This grouping of genes to identify sets of genes that appear to be differentially expressed between aRNA and mRNA resulted in two clusters (91 elements among clusters 5 and 6) that have the largest difference between aRNA and mRNA (Figure 6).

Figure 5. K- means cluster analysis of the 6912 elements using a userdefined cluster number of 6. 45 iterations were carried out to group the genes within a given cluster using a data centroid based search. The total score was calculated to be 1.082e+004. The most closely clustering genes had a similarity value of 0 and the least similar gene had a similarity value of 1.798e+308.

Figure 6. An analysis of variance calculation of K-mean clusters 5 and 6. This plot indicates the confidence limit of the data. A p-value, of less than 0.0001 was used, indicating that the genes represented in this plot are unique at the 99.9999% cut-off value.

Analysis of variance (ANOVA) of genes in clusters 5 and 6 indicated that the clusters contained genes that behave distinctly between the mRNA and aRNA samples at a confidence limit of 99.99999% (p<0.00001). ANOVA measurements processed the gene-by-gene fluctuations from the mean value and accounted for variance across samples.

A scatter plot analysis of the raw Cy3 and Cy5 values of all the 6912 elements within the 5 gene clusters is shown in Figure 7 (4 plots). The top two plots represent all the elements and the bottom two depict the genes that show the largest differences in signal. Most of the genes that are distinct between the samples are expressed at lower levels (low fluorescent signal). These differences were more exaggerated in the aRNA than the mRNA sample because the signal-to-noise ratio was typically much greater in the aRNA sample. The distinct genes in the aRNA panel might be elements that were not clearly discernible in the mRNA sample due to ribosomal contamination (27% in the mRNA used for this analysis). The presence of ribosomal RNA can increase background in mRNA samples, resulting in variations in mRNA concentration between samples and decreasing the efficiency of cDNA probe synthesis. Thus the presence of ribosomal RNA could have cumulatively skewed the detection and quantification of genes that were expressed in very low amounts when mRNA or total RNA was used.

Figure 7. Scatter Plot Analysis of all Array Elements. A scatter plot of the Cy5 vs. Cy3 values obtained for an aRNA and a mRNA array is shown. The top two panels depict the 6 clusters (obtained after K-Mean Clustering Analysis) containing all 6912 elements. A subset of elements that are distinct between the two arrays and which deviate the most in signal intensity are depicted in the lower panels.

Amplification of RNA thus provides a means of measuring expression from genes transcribed at very low levels. In many cases the RNA concentration of an experimental sample is under the optimal required amount for synthesizing labeled cDNA for microarray analysis. MessageAmp is a viable technology for increasing the yield of useful probe and can greatly lower the starting amount of RNA required to produce biologically relevant signals.

More on Microarray Analysis
In future columns we will continue to report results from MessageAmp microarray studies from Ambion researchers, our collaborators, and our customers. If you have results you would like to share, please contact Lakshmi Madabusi at or call 1-800-888-8804 x6308.


1. Quakenbush J (2001). Computational analysis of microarray data. Nature Reviews Genetics. 2(6): 418427.

2. Tavazoie S, Hughes JD, Campbell MJ, Chos RJ and Church GM (1999). Systematic determination of genetic network architecture. Nature Genetics. 22: 281285.

Cy3 is a trademark of Amersham Biosciences.

back to top

Ordering Information
For prices and availability, please contact our Customer Service Department.
Cat# Product Name Size 1705 Amino Allyl cDNA Labeling Kit 15 rxns 1750 MessageAmp aRNA Kit 20 rxns


Page: All 1 2 3 4 5 6 7

Related biology technology :

1. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
2. Epitope-Tagging Vectors for Functional Analysis in Yeast
3. Map and Link Human Genetic Disorders with SSLP Analysis
4. CastAway Precast Gels for Rapid Automated DNA Sequence Analysis
5. Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf cMaster RTplusPCR System
6. Structural Analysis of Glycosylated Peptides in Complex Mixtures with Ion Trap MSn
7. Use of Transcriptor Reverse Transcriptase in Microarray Analysis
8. Increased Sensitivity in Microarray Analysis
9. Gene-Expression Analysis of TP, DPD, and TS Using the LightCycler mRNA Quantification KitsPLUS
10. Agilent 2100 Bioanalyzer Automated Analysis System
11. aRNA Synthesis and Labeling for Array Analysis
Post Your Comments:
TAG: Analysis Microarray Data

(Date:7/14/2014)... CA (PRWEB) July 14, 2014 ... patient registry platform, announced that three PatientCrossroads-based ... dystrophies; the Phelan-McDermid Syndrome International Registry; and ... Syndrome—have been awarded contracts by the Patient-Centered ... the creation of a new health data ...
(Date:7/13/2014)... discovery 30 years ago of soccer-ball-shaped carbon molecules called ... Now, there appears to be a new ball on ... and Tsinghua University in China have shown that a ... cage similar to a carbon buckyball. It,s the first ... matter of speculationdoes indeed exist. , "This is the ...
(Date:7/13/2014)... Dallas, TX (PRWEB) July 13, 2014 ... Silver Industry” is a professional and in-depth study on ... Global and China. , The report provides a basic ... chain structure. Global market analysis and Chinese domestic market ... trends and competitive landscape of the market. A comparison ...
(Date:7/12/2014)... York (PRWEB) July 12, 2014 ... for various reagents used in the life ... driving the life science reagents market towards ... include manufacturers and providers of life science ... Market Report: . , The ...
Breaking Biology Technology:PatientCrossroads Fuels Development of New National Clinical Research Network 2PatientCrossroads Fuels Development of New National Clinical Research Network 3Researchers discover boron 'buckyball' 2Researchers discover boron 'buckyball' 32014 Nano Silver Industry in Global and China Regions Development Proposals Analysis Available at 22014 Nano Silver Industry in Global and China Regions Development Proposals Analysis Available at 3Transparency Market Research: Life Science Reagents Market- PCR Reagent Kits, Cell and Tissue Culture, Flow Cytometry, Electrophoresis and Chromatography 2Transparency Market Research: Life Science Reagents Market- PCR Reagent Kits, Cell and Tissue Culture, Flow Cytometry, Electrophoresis and Chromatography 3
... SAN FRANCISCO, May 22 Takeda San ... Haak-Frendscho, Ph.D., as president,and chief scientific officer. ... activities at Takeda San Francisco including overall ... operations for,antibody therapeutics., Takeda San Francisco ...
... ... Emerging Pattern, NORTH BERGEN, N.J., ... Friday, 20 people arrived, one by one, for emergency care at,the ... nausea and diarrhea., A coincidence? Or the harbinger of a ...
... AstraZeneca (NYSE:,AZN) announced today that it supports ... the company,s latest step to provide the public,with ... Sponsored by Sens. Charles Grassley of Iowa ... a national registry of payments that,biopharmaceutical companies, device ...
Cached Biology Technology:Takeda's Antibody Therapeutics Research Company, Takeda San Francisco, Inc. Appoints Mary Haak-Frendscho, Ph.D. as President and CSO 2E. coli Outbreak in New Jersey? 2E. coli Outbreak in New Jersey? 3AstraZeneca Supports Transparency Legislation on Payments to Health Care Providers 2
(Date:7/14/2014)... The ability to reliably and safely make in the laboratory ... is one key step closer to reality. , Writing today ... by University of Wisconsin-Madison stem cell researcher Igor Slukvin reports ... stem cells and turning them into both red and the ... The research is important because it identifies how nature itself ...
(Date:7/13/2014)... A new study in women suggests that experiencing one ... single high-fat meal can slow the body,s metabolism, potentially ... about the previous day,s stressors before giving them a ... fat. The scientists then measured their metabolic rate ... and fat and took measures of blood sugar, ...
(Date:7/13/2014)... French . , ... led by the Research Institute of the McGill University ... oral medication is showing significant progress in restoring vision ... this inherited retinal disease that causes visual impairment ranging ... The study is published today in the scientific journal ...
Breaking Biology News(10 mins):Wisconsin scientists find genetic recipe to turn stem cells to blood 2Wisconsin scientists find genetic recipe to turn stem cells to blood 3Weighty issue: Stress and high-fat meals combine to slow metabolism in women 2Weighty issue: Stress and high-fat meals combine to slow metabolism in women 3Weighty issue: Stress and high-fat meals combine to slow metabolism in women 4MUHC researcher unveils novel treatment for a form of childhood blindness 2
... Mich. A patented Michigan State University process ... means extra nutrients don,t have to be added, ... The AFEX (ammonia fiber expansion) pretreatment process, ... chemical engineering and materials science, uses ammonia to ...
... major threats to biodiversity and in many cases they ... their effective management it is important to understand which ... being invaded. The first map of the level ... in the Wiley-Blackwell journal Diversity and Distributions . ...
... the large tectonic plates and the superimposed continents is ... the Earth,s mantle, but rather retroacts on this internal ... a thermal blanket, which leads to an accumulation of ... break-up of the super-continents. , These results of ...
Cached Biology News:Process can cut the cost of making cellulosic biofuels 2The continents as a heat blanket 2
... Services include: ,ICC and IHC Protocol ... Analyses ,Antibody Screenings in Animal or ... Labeling of Cells and Tissues ,Peroxidase, ... ,Light, Fluorescent, or Electron Microscopy ,Protein ...
Use our experts and resources to develop reagent systems for your applications....
Request Info...
Request Info...
Biology Products: