Microarray and dna chips for transcriptome study


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Microarray and dna chips for transcriptome study

  1. 1. Microarray and DNA chips for Transcriptome study Amna Siddiqui,Areeba Khan, Faiq Sabih, Hammad Naveed, Hassan Fajri BBS-6
  2. 2. Overview • Transcriptome study; why needed? • Methods used • What are Microarray and DNA chips? • Difference between the two • Method used to study one or more transcriptome • Complications • Alternate method to compare two transcriptomes • Yeast transcriptome • Applications
  3. 3. Transcriptome A transcriptome is the collection of all the mRNA transcripts transcribed from the DNA (genome), at any one time, in a particular cell. • 4% of the total RNA content of the cell • Specifies the composition of the proteome (concerned with coding regions that will be expressed) • Expression triggered by environmental factors • If conditions are not optimum, total expression can be switched off.
  4. 4. Transcriptome study • Study of the transcriptome of any organism’s cell is also called expression profiling • By analyzing transcriptome sequence, it can be determined when and where a gene is turned off or on in a particular cell • Organisms may have same genes but different gene expression leading to difference in behaviours • Thus, comparison of two transcriptomes (from different specie or different cell types) can be used to determine properties of each cell type • Doing transcriptome study of a diseased or cancerous cell can lead to information about the gene responsible for producing abnormality (gene expression less or more)
  5. 5. Methods used in Transcriptome Study Serial Analysis of Gene Expression (SAGE) Massively parallel signature sequencing (MPSS) Microarray and DNA chips RNA-Seq (emerging method)
  6. 6. Microarray and DNA chips • Microarray method used to check gene expression • Microarrays or DNA chips use a thin glass microscopic slide, silicon chip or nylon membrane • Thousands of reference genes can be immobilized, spotted or synthesized in situ on a small space on these glass slides • Works on principle of hybridization of mRNA (converted to cDNA) of concerned cell with immobilized cDNA/oligonucleotide sequence present on array slide
  7. 7. Microarray and DNA chips • Advantage over SAGE: rapid evaluation of the comparison of two transcriptomes can be achieved by running them simultaneously on identical arrays and checking hybridization patterns of the two • Further refined results can be achieved by using mRNA that is bound to ribosome in cell. Ribosome-bound mRNA gives part of transcriptome that is actively being used in protein synthesis
  8. 8. Difference between two Microarray • Uses PCR products or cDNA of the genes of interest • Spotted on the surface of the glass slide or nylon membrane DNA chip • The oligonucleotides match positions within the gene of interest • Mixture of oligonucleotides synthesized in situ on the surface of the glass slide or silicon membrane.
  9. 9. Objectives of Microarray and DNA chips • Two major objectives Objective: Identify gene whose mRNA is present Microarray: PCR products or cDNA derived from all genes of interest DNA chip: Mixture of oligonucleotides synthesized which match positions in the gene
  10. 10. Objectives of Microarray and DNA chips Objective: Determine relative amounts of mRNA present Each position in array contains up to 109 copies of probe molecules. Signal intensity at each position will determine level of hybridization of probe with mRNA (high copy number, more hybridization)
  11. 11. Basic Flowchart of Transcriptome study using Microarray/DNA chip Collect mRNA molecules from a cell Use Reverse Transcriptase (RT) enzyme to produce cDNA molecules from the mRNA Label cDNA with flourescent dyes Prepare microarray/DNA chip (cDNA from reference genes or oligonucleotide mixture) Place labeled cDNA on microarray slide Hybridization of labeled cDNA with cDNA (complimentary) on microarray Larger mRNA amount in cell (more expression), more hybridization.Vice versa Scan array slide. More flourescence, more intensity of expression
  12. 12. Microarray and DNA chip method
  13. 13. Microarray/DNA chip after hybridization. Color intensity shows level of hybridization.The cDNA prepared from mRNA is first labeled with fluorescent marker (like Cy3 and Cy5), then hybridized with array to produce such a pattern.
  14. 14. Complications • Hybridization analysis will have insufficient specificity to distinguish between every mRNA that could be present. Two mRNAs, similar sequences, may cross- hybridize with each other’s specific probe on the array Paralogous genes active in same tissue – group of related mRNAs can hybridize with members of the same gene family Distinguishing which specific mRNA is present and how much is present becomes difficult Two or more different mRNAs could have been derived from same gene – alternate splicing concept
  15. 15. Complications • Alternate splicing
  16. 16. Complications • When comparing more than one transcriptome, differences in mRNA amount and hybridization intensities must be due to difference in transcripts rather than due to experimental errors. • Experimental errors could include: – Amount of target DNA on array – Efficiency with which probe has been labeled – Effectiveness of hybridization process • Absolute precision and exact reproducibility of results is almost impossible in different laboratories doing same analysis (due to these experimental factors)
  17. 17. Normalization Procedures to Counter Experimental Factors • To counter these experimental factors, certain normalization procedures are employed. • Enables results from different array experiments to be accurately compared • Normalization procedure: – Negative controls, so that background can be determined in each experiment – Positive controls, always give identical signals
  18. 18. Normalization Procedures to Counter Experimental Factors • In vertebrates, actin gene is used as positive control – Its expression level is fairly constant in any particular tissue – Even in developmental, or diseased state
  19. 19. Alternative method to study and compare two transcriptomes • Design experiment alternately • Compare two transcriptomes directly, on single array • Label the cDNA with different flourescent probes • Scan the array at different wavelengths of light • Determine relative intensities of the two types fluorescence at each position • Differences in the mRNA content of the two transcriptomes can be directly analyzed.
  20. 20. Yeast transcriptome: An example • Ideally suited for transcriptome studies • Little changes in yeast transcriptome, if biochemical environment is constant • Glucose rich – stable. Glucose depletion: causes corresponding restructuring of transcriptome. • Transcriptome also undergoes restructuring during cellular differentiation. • Sporulation pathway.
  21. 21. Yeast transcriptome: An example • Spores adapt its mRNA at each stage to the changing stressful conditions. • Acts as model organism to study interactions between genome and environmental signals in higher eukaryotes. • Transcriptome studies help to annotate a genome sequence, helping in identifying gene functions.
  22. 22. Applications • Studying the transcriptome can lead to various applications – Transcriptomes of stem cell and cancer cells can be studied by researchers to understand cellular differentiation and carcinogenesis – Transcriptomes of human oocytes and embryos can be studied to understand molecular mechanisms and signaling pathways in embryonic development – Used in biomarker discovery
  23. 23. Thank you!