Transcriptomics(Microarray: Chip and Image Analysis).pptx
- 2. Introduction • Transcriptomics is the study of the ‘transcriptome’ has been attributed to Charles Auffray. • Transcriptome is now widely understood to mean the complete set of all the RNA molecules expressed in any given entity, such as a cell, tissue, or organism at a given time. • Transcriptomics covers all types of transcripts, including mRNAs, miRNAs, and different types of lncRNAs. • In contrast with the genome, which is characterized by its stability, the transcriptome actively changes. In fact, an organism's transcriptome varies depending on many factors, including stage of development and environmental conditions.
- 3. Introduction • Transcriptomics encompasses everything relating to RNAs. This includes- • Transcription and expression level • Function, location • Trafficking, degradation • Structures of transcripts and their parent genes with regard to start sites, 5 and 3 end ′ ′ sequences, splicing patterns, and • Posttranscriptional modifications • Modern transcriptomics uses high-throughput methods to analyze the expression of multiple transcripts in different physiological or pathological conditions. • This is rapidly expanding our understanding of the relationships between the transcriptome and the phenotype across a wide range of living entities.
- 4. Enabling Technologies in Transcriptomics • Expressed Sequence Tags (ESTs) • Serial Analysis of Gene Expression (SAGE) • Microarray • RNA Sequencing
- 5. Isolation of RNA • All transcriptomic methods require RNA to first be isolated from the experimental organism before transcripts can be recorded. • Although biological systems are incredibly diverse, RNA extraction techniques are broadly similar and involve the following steps: a. Mechanical disruption of cells or tissues, b. Disruption of RNase with chaotropic salts (lithium chloride & magnesium chloride), c. Disruption of macromolecules and nucleotide complexes, d. Separation of RNA from undesired biomolecules including DNA, and e. Concentration of the RNA via precipitation from solution • Isolated RNA may additionally be treated with DNase to digest any traces of DNA. • It is necessary to enrich messenger RNA as total RNA extracts are typically 98% rRNA. Enrichment for transcripts can be performed by poly-A affinity methods. • Snap-freezing of tissue prior to RNA isolation is typical, and care is taken to reduce exposure to RNase enzymes once isolation is complete.
- 6. Expressed Sequence Tag (EST) • An EST is a short nucleotide sequence (100-800 nt long) generated from a single RNA transcript. • RNA is first copied as cDNA by a reverse transcriptase enzyme before the resultant cDNA is sequenced. Thus, an EST is a short sub-sequence of a cDNA sequence. • The Sanger method of sequencing was predominant until the advent of high- throughput methods such as sequencing by synthesis. • Because ESTs don't require prior knowledge of the organism from which they come, they can also be made from mixtures of organisms or environmental samples. • ESTs may be used to identify gene transcripts, and were instrumental in gene discovery and in gene-sequence determination • EST approaches have largely been superseded by whole genome and transcriptome sequencing and metagenome sequencing.
- 7. EST
- 8. SAGE • SAGE was a development of EST methodology to increase the throughput of the tags generated and allow some quantitation of transcript abundance. • cDNA is generated from the RNA but is then digested into 11 bp “tag” fragments using restriction enzymes that cut at a specific sequence, and 11 base pairs along from that sequence. • These cDNA tags are then concatenated head-to-tail into long strands (>500 bp) and sequenced using low-throughput, but long read length methods such as Sanger sequencing. • Once the sequences are deconvoluted into their original 11 bp tags, they can be used to find the frequency of each tag. The tag frequency can be used to report on transcription of the gene that the tag came from. • If a reference genome is available, these tags can sometimes be aligned to identify their corresponding gene. • If a reference genome is unavailable, the tags can simply be directly used as diagnostic markers if found to be differentially expressed in a disease state. • SAGE methods produce information on more genes than was possible when sequencing single ESTs, but the sample preparation and data analysis are typically more labor intensive.
- 9. SAGE
- 10. EST Vs SAGE • SAGE was 26 times more sensitive than the EST method in detecting these transcripts • EST target individual transcript whereas SAGE target multiple target • Tag size of EST is 100-800 nt long and in SAGE it is 11bp long • SAGE is labor intensive and require bioinformatical tools also
- 11. Microarray • A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time. • DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene. • Often, these slides are referred to as gene chips or DNA chips. • The DNA molecules attached to each slide act as probes to detect gene expression, which is also known as the transcriptome or the set of messenger RNA (mRNA) transcripts expressed by a group of genes.
- 12. Microarray • mRNA molecules are typically collected from both an experimental sample and a reference sample. For example, the reference sample could be collected from a healthy individual, and the experimental sample could be collected from an individual with a disease like cancer. • The two mRNA samples are then converted into complementary DNA (cDNA), and each sample is labeled with a fluorescent probe of a different color. For instance, the experimental cDNA sample may be labeled with a red fluorescent dye, whereas the reference cDNA may be labeled with a green fluorescent dye. • The two samples are then mixed together and allowed to bind to the microarray slide. The process in which the cDNA molecules bind to the DNA probes on the slide is called hybridization. • Following hybridization, the microarray is scanned to measure the expression of each gene printed on the slide. If the expression of a particular gene is higher in the experimental sample than in the reference sample, then the corresponding spot on the microarray appears red. In contrast, if the expression in the experimental sample is lower than in the reference sample, then the spot appears green. Finally, if there is equal expression in the two samples, then the spot appears yellow. • The data gathered through microarrays can be used to create gene expression profiles, which show simultaneous changes in the expression of many genes in response to a particular condition or treatment.
- 13. Microarray
- 14. Steps in Microarray Analysis
- 15. Microarray: Chip and Image Analysis
- 16. Application of Microarray Technology CGH: Comparative Genome Hybridization
- 18. Microarray Application in Drug Discovery
- 19. RNA Sequencing • RNA sequencing (abbreviated as RNA-Seq) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing cellular transcriptome. • Compared to previous Sanger sequencing- and microarray-based methods, RNA-Seq provides far higher coverage and greater resolution of the dynamic nature of the transcriptome. • Beyond quantifying gene expression, the data generated by RNA-Seq facilitate the discovery of novel transcripts, identification of alternatively spliced genes, and detection of allele-specific expression.
- 20. Steps in RNA Sequencing • RNA isolation • RNA selection/enrichment/depletion • cDNA synthesis • Fragmentation and size selection • High-throughput cDNA sequencing • Small RNA and other non-coding RNA sequencing • Align the sequence reads to a reference genome if available or assembled de novo to produce an RNA sequence map that spans the transcriptome
- 21. RNA Sequencing
- 22. RNA Sequencing