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 lmadabusi@ambion.com or call 1-800-888-8804 x6308.


REFERENCES

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
'"/>

Source:


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:
*Name:
*Comment:
*Email:
TAG: Analysis Microarray Data

(Date:8/21/2014)... , Aug. 21, 2014 Telomere ... of their Nutraceutical "TELO-20 for Dogs" with ... Telomere-lengthening supplement in the world for dogs. Telomeres ... every chromosome in the body. A wealth of ... experts in Telomere Science and Aging, including Nobel ...
(Date:8/21/2014)... HONG KONG , Aug. 21, 2014  China Cord ... today announced its plan to release financial results for ... August 28, 2014, after market close in the US.  ... call at 8:00 a.m. ET on Friday, August 29, ... brief overview of the Company,s recent developments, followed by ...
(Date:8/21/2014)... (PRWEB) August 21, 2014 ... has been recognized as one of the nation’s ... 5000 magazine for the 2nd consecutive year. Inc. ... 33rd annual Inc. 500|5000, an exclusive ranking of ... the most comprehensive look at the most important ...
(Date:8/20/2014)... the first time, chemists have succeeded in measuring vibrational ... resolution. The study reveals how vibration of a single ... , The study was performed at the University ... the University of Jyvskyl works as a visiting fellow ... the study. The second team was lead by Professor ...
Breaking Biology Technology:Telomere Biosciences Announces: TELO-20 for Dogs, the World's First Telomere-Lengthening Supplement for Dogs, to Promote Healthy Aging and Longevity 2Telomere Biosciences Announces: TELO-20 for Dogs, the World's First Telomere-Lengthening Supplement for Dogs, to Promote Healthy Aging and Longevity 3China Cord Blood Corporation to Report First Quarter of Fiscal 2015 Financial Results 2SoundConnect Returns on Inc. 5000 List of Fastest Growing Companies 2SoundConnect Returns on Inc. 5000 List of Fastest Growing Companies 3Seeing a molecule breathe 2
... Wis. Ten $1,000 scholarships and a top prize ... young entrepreneurs in a business-plan competition sponsored by BizFilings ... Entrepreneur Foundation . , ,The contest, named Plan for the ... until May 31. They'll be judged by an independent panel. ...
... unseen battle now raging in the workplace. It pits incumbent ... who covet the traditional employment deal money, career progression ... playing the organizational game by traditional rules, e.g., long hours ... to do whatever the business requires. Only Type A's need ...
... Mirus Bio Corporation says its scientists have ... cheaply. , ,Antibodies, naturally part of the immune system, ... for diagnostics or research. Mirus' new technique, described in ... into animals, whose immune systems produce the antibodies that ...
Cached Biology Technology:Who will win the battle to redefine the workplace? 2Who will win the battle to redefine the workplace? 3Who will win the battle to redefine the workplace? 4Who will win the battle to redefine the workplace? 5
(Date:8/21/2014)... In a finding that has implications for life ... elsewhere in the solar system, LSU Associate Professor of ... the National Science Foundation, or NSF, this week published ... a lake that lies 800 meters (2600 feet) beneath ... "viable microbial ecosystems.", Given that more than 400 subglacial ...
(Date:8/21/2014)... A study recently published in the journal ... shows a new role for the protein adenomatous polyposis ... cause of cancer-related deaths in the U.S. , Lead ... Molecular Biosciences and co-leader of the Cancer Biology program ... part of her career trying to understand the various ...
(Date:8/21/2014)... North Carolina State University have developed a novel and ... has applications for creating new materials as well as ... new technique allows us to model much larger and ... more quickly," says Nan Li, lead author of a ... NC State,s Department of Materials Science and Engineering. "This ...
Breaking Biology News(10 mins):800 meters beneath Antarctic ice sheet, subglacial lake holds viable microbial ecosystems 2800 meters beneath Antarctic ice sheet, subglacial lake holds viable microbial ecosystems 3800 meters beneath Antarctic ice sheet, subglacial lake holds viable microbial ecosystems 4Research offers insight into cellular biology of colorectal cancer 2Research offers insight into cellular biology of colorectal cancer 3Researchers develop models to study polyelectrolytes, including DNA and RNA 2
... debate of nature versus nurture continuesthis time in what ... Journal of Communication found that a specific ... who engaged in increased viewing of violent TV and ... Vossen, and Patti Valkenburg of the University of Amsterdam,s ...
... are probably best-loved insects. As such, they are relatively ... of the country are explored most thoroughly. First eastern ... modern taxonomy Carl Linnaeus himself, over 250 ... centuries, naturalists have been cataloguing species diversity of eastern ...
... The UK could generate almost half its energy needs from ... by 2050, new research suggests. Scientists from the Tyndall ... found that the UK could produce up to 44% of ... The study, published in Energy Policy journal, ...
Cached Biology News:Genetics linked to children viewing high amounts of violent media 2Two new butterfly species discovered in eastern USA 2UK failing to harness its bioenergy potential 2
... Streptavidin-Horseradish Peroxidase Conjugate, ... to reporter enzyme molecules ... and in situ hybridization ... Blotting & Labeling & ...
normal rat IgM AF647...
Animal Origin Free...
IHC Detection Kit (Broad Spectrum AEC)....
Biology Products: