L10 Transcriptome Flashcards
Transcriptome (general)
Genomic information transcribed into RNA
Complex, contain copies of hundred-thousands of different mRNAs
Genes may produce different types of mRNA, hence more complex and dynamic than the genome
Transcriptome is variable, genome is stable.
Analysis provides a view of when and where a gene is switched on or off in various types of cells and tissues.
Cell specific expression of genes
Analysing the Transcriptome
Using northern blot, RT-PCR, Q-PCR
studies only a few genes at a time
human genome 30,000
MICROARRAYS - looks at many genes
Microarray
Set of probes (short oligonucleotides) that have been immobilized onto a surface (glass slide), generating a DNA chip.
Each probe represent a specific gene, and high density arrays contain all gene of a given genome
Position of each probe is known.
Types of microarray
Spotted arrays- cDNA, PCR products or oligonucleotides are spotted by a robot onto the surface of the glass slide at a precise location. DNA must be denatures to convert into single strand.
Main disadvantage: one gene, one probe.
Leads to cross hybridization and failure to specifically detect some transcripts.
Type2: In situ oligonucleotide array
Synthesized single stranded oligos (25bp)
one probe, several gene
Microarray overview
Based on nucleic acid hybridization
RNA extraction > cDNA synthesis > labelling
Labels used are fluorescent.
Hybridisation and detection
labelled cDNA > mix and denature to obtain single strand labelled cDNA. cDNA will compete. Hence it will enable identification which gene is expressed.
Low level analysis:
difference in expression levels of a gene between experimental treatments. e.g treatment 1 intensity : 3000
treatment 2 intensity: 1500. Hence, 3000/1500 = 2
2x expression increase of gene in treatment 1.
High level analysis:
Cluster analysis: grouping of genes that respond similarly to experimental treatments.
Dendograms are then used to “group” genes. implying that they may have similar functions.
Application of microarray
Generate a view of overall gene expression patterns
Find targets of regulatory genes
Identification of genes whose expression is associated to a physiogical condition
Study of disease-comparison of healthy and unhealthy tissue > Useful for diagnostic marker
Pharmacogenomics: How genetic composition affects response to drugs - personalized medicine.
Disadvantages of microarrays
Based on hybridisation
Depends on genome sequence knowledge
Non specific binding or cross hybridisation
Mismatches in sequences from different strains
Complex normalisation
RNA - Seq as an advantageous alternative.
BAsed on high throughput sequencing (illumina)
Comvert all mRNA into cDNA followed by massive parallel sequencing
No need for genome sequence
Maps transcription starts and SNPs broad detection range
Outline RNA-seq
RNA and mRNA isolation
Convert into cDNA
Adapter ligation to cDNA and sequencing (Illumina)
Align sequences obtained > plot graph
Pros: More reads obtained, more expressed.
