Microarray I and II Flashcards
What is a transcriptome?
A complete set of the transcripts encoded from the genome
What can the transcriptome tell you?
Genes that exhibit cell and tissue specific expression
What can a transcriptome tell you about a disease?
The molecular basis behind it
How can an unknown drug impact genes?
Genes that are expressed in response to environmental toxins or drugs
What are genes expressed in response to?
Pathogens, diseases, and any mode of infection
What is northern blotting?
This is a method od visualizing and detecting RNA transcripts
What is western blotting?
A method of detecting proteins
What is southern blotting?
A method of detecting DNA
What is the goal of a DNA microarray?
To detect which genes are turned on and off depending on the environment
What does the chip contain?
- Many ss probes
- Fluorescent-labelled mRNAs or cDNAs to bind to the probe
- The fluorescent intensity tells us the mRNA levels binding to probes
How is the probe design specific?
It does not allow for cross hybridization
How is the probe design homogeneous?
Binds to complementary DNA at the same melting temperature
How is the probe design sensitive?
Does not form secondary structures that could interfere with the hybridization between the probe and mRNA
What is the microarray procedure?
- Isolate mRNA
- Reverse transcribe to cDNA
- Label with Cy3 (green) and Cy5 (red)
What is the agilent method?
Ink-jet print-head uniformly deposits small, accurate volumes of nucleic acids which builds the oligonucleotide probes one base at a time
What is the affymetric method?
The probes are synthesized on wafer and a photomask covers the the silicon wafer and light shines through the holes the light removes blocking compounds which prevents the base addition then you flood with a base (A, T, C, or G) that binds to the uncovered area and continue
How do you label the nucleic acids?
- Random priming of dsDNA
- Directly labeling the mRNA with fluorescent molecules
- Poly-T primed cDNA
- Amplification by transcription
What are Cy3 and Cy5?
Water-soluble fluorescent dyes of the cyanine family that is excited by green and red lasers respectively
To quantify the segmentation which central tendency should you use?
Median: it is less likely to be skewed compared to the mean it is robust and tells you the outliers
Why do we normalize the data?
We do this so we can compare the magnitude of up and down regulation equally rather than treating the quantity as less 2 fold Vs. 1/2
What is an MA plot?
It shows the intensity dependent effect on log expression ratios
What is differential expression?
Genes that are turned on or rather have a higher or lower than normal expression
How do we identify the genes that are differentially expressed?
We use a cut-off value
What is clustering?
Grouping data based on similiarities which makes it easier to analyze
What can the similarities be based on?
- Euclidean distance - Distance between 2 points
- Correlation distance - Similarity of the diretions in which 2 vectors point
What is hierarchichal clustering?
Clustering based on similarity
What is partitional clustering?
When you cluster based on K groups
What is agglomerative clustering?
Start with a single gene and build up to a super-cluster
What is divisive clustering?
Start with a group of genes and build to a single gene
What is 2D clustering?
This organizes rows and columns of genes together so you can determine co-regulated genes
What is an ORF microarray?
Probing for sequence of known genes about 10 nucleotides at a time
What is tiling microarray?
Tile the entire genome with probes rather than just the coding sequence
What do longer UTR regions tell us?
That the gene undergoes more gene regulation
What do shorter UTR regions tell us?
Reduced need for regulation
