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:7/30/2015)... ... 30, 2015 , ... The 2015 Market Research Report on the Global ... the global Propanol market with a focus on the Chinese situation. Major companies included ... Oxea, ISU Chemical, Sanofi, Royal Dutch Shell, LG Chem and Zhejiang Xinhua Chemical. , ...
(Date:7/30/2015)... ... July 30, 2015 , ... Leading regenerative veterinary ... of its canine osteoarthritis stem cell product, currently under development for FDA approval. ... will be marketed in the US by Aratana. This product, termed AT-016, is ...
(Date:7/30/2015)... --  Senomyx , Inc. (NASDAQ: SNMX ), a ... develop, and commercialize novel flavor ingredients for the food, ... for the second quarter 2015. "Moving ... track to achieve our commercial and financial goals," stated ... Company. "Since our last quarterly earnings report, our two ...
(Date:7/30/2015)... COPENHAGEN, Denmark , July 30, 2015 /PRNewswire/ ... a biotechnology company that applies its innovative TransCon ... announced positive top-line results from a six-month Phase ... of once-weekly TransCon Growth Hormone in 53 treatment-naïve, ... "We are extremely pleased with the ...
Breaking Biology Technology:Global Propanol Market Technical Data and Manufacturing Analysis 2020 2Global Propanol Market Technical Data and Manufacturing Analysis 2020 3VetStem Biopharma Successfully Completes Large Milestone Efficacy Study for its Flagship Canine Osteoarthritis Stem Cell Product 2VetStem Biopharma Successfully Completes Large Milestone Efficacy Study for its Flagship Canine Osteoarthritis Stem Cell Product 3SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 2SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 3SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 4SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 5SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 6SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 7SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 8SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 9Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 2Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 3Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 4Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 5Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 6Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 7
... officials announced today a five-year agreement with K.S.R. Educational ... training in nanomedicine and nanotechnology. "We are very ... 16 institutions of higher education in Tamil Nadu, India," ... has the same commitment to deliver quality education to ...
... Corp. (NASDAQ: KOOL ), a leading supplier ... adult stem cells, said today that its submission for ... to concentrate stem cells from cord blood, has been ... in China. This represents the successful completion of product ...
... Corporation (NASDAQ: UTHR ) (the "Company") today announced ... conditions, $210 million aggregate principal amount of convertible senior notes ... 144A under the Securities Act of 1933, as amended (the ... initial purchaser of the notes an option to purchase up ...
Cached Biology Technology:MU signs academic agreement with K.S.R. Educational Institutions of India 2ThermoGenesis Announces Acceptance of AXP® Submission by Regulatory Body in China 2ThermoGenesis Announces Acceptance of AXP® Submission by Regulatory Body in China 3United Therapeutics Announces Proposed Private Offering of $210 Million of Convertible Senior Notes and Authorization of Stock Repurchase Program 2United Therapeutics Announces Proposed Private Offering of $210 Million of Convertible Senior Notes and Authorization of Stock Repurchase Program 3United Therapeutics Announces Proposed Private Offering of $210 Million of Convertible Senior Notes and Authorization of Stock Repurchase Program 4
(Date:6/24/2015)... 2015 The biologics safety testing market is ... the market include growth of the pharmaceutical and biological ... Over the years, the number of drug approvals in ... the FDA approved 225 drug applications and Health Canada ... Europe accounted for 3,822 of the pharmaceutical ...
(Date:6/24/2015)... SAN JOSE, Calif. , June 24, 2015 ... leading developer of human interface solutions, today announced ... fingerprint sensor to provide secure authentication for its ... Having recently reached more than 200 million shipments ... shipment with Sharp reinforces Synaptics, strength, scalability and ...
(Date:6/24/2015)... 2015 Biometry authentication provider KeyLemon today ... solution one face in. Logo ... face in, entering the expanding biometric authentication market ... biometric answer to the password problem. With advanced ... with KeyLemon, one face in allows users to ...
Breaking Biology News(10 mins):Biological Safety Testing Market by Application, Product, & Test - Global Forecast to 2019 2Biological Safety Testing Market by Application, Product, & Test - Global Forecast to 2019 3Biological Safety Testing Market by Application, Product, & Test - Global Forecast to 2019 4Synaptics Brings Natural ID Technology to Sharp's Newest Flagship Smartphone 2KeyLemon Announces Partnership With Biometric Password Solution onefacein 2
... pathogens deploy effector proteins as part of their ,molecular ... New research has revealed the structure of a bacterial ... host. , Gram-negative bacterial pathogens can deliver effector proteins ... to manipulate host cell processes for the benefit of ...
... - Scientists at the U.S. Department of Energy,s Brookhaven ... mechanism plants use to limit the production of fatty ... biochemical pathways to increase the production of plant oils ... "Now that we understand how this system operates ...
... diversity is increasingly threatening Mother Nature,s ability to provide humans ... and protection from pests and disease, according to a sweeping ... of ecologists, including biologists from the University of British Columbia ... the June 7 edition of the journal Nature ...
Cached Biology News:The activity of a bacterial effector protein seen in molecular detail 2Scientists identify mechanism for regulating plant oil production 2Scientists identify mechanism for regulating plant oil production 3Loss of biodiversity increasingly threatens human well-being: UBC, University of Michigan research 2
...
... Human CD66 acd Recognizes human ... neutrophils and cells in some cases ... with transfectants containing BGPa, NCA and ... Immunogen: Human ...
Request Info...
... GOAT ANTI-MOUSE IgG+IgA+IgM (H+L) ... with highly purified horseradish ... antibody has been absorbed ... fetal bovine, horse, goat, ...
Biology Products: