Microarray and RNA-seq

What are the general procedures for running a glass array/DNA microarray?

1. 2 growth conditions exist, one labeled with Cy3, and the other with Cy5. 2. Mix and hybridize to microarray 3. 3 different colors expressed: only condition 1, conditions 1 and 2, and only condition 2

What are the types of background subtraction?

1. Background from local area around each spot 2. Spots with lowest signal intensities as background 3. Background from blank areas (no nucleic acids)

How does Illumina sequencing work?

1. DNA first attached to primers on a slide and amplified so that local colonies are formed (bridge amplification) 2. One type of nt is added at a time, non-incorporated nt are washed 3. Camera used to take images of the fluorescently-labeled nt and dye is chemically removed from the DNA (reversible dye-terminators), allows for next cycle

What are attributes of affymetrix genechips?

1. Probes are 25-mer oligos synthesized in situ using a photolithographic approach. 2. At least 5 oligos/gene are used + an equal number of controls. 3. Each pair has two oligos- perfect match (PM) and mismatch (MM, one base change). 4. The scanned result for a gene is the average difference between PM and MM signals. 5. Only a single fluorochrome is used per hybridization

What techniques are used for microarray data analysis?

1. Scatter plot 2. K-means clustering

How does construction of a cDNA microarray occur?

1. Starts with normalized cDNA library 2. EST clones are sequenced 3. Clones are re-racked (using robotics) 4. Unique EST clones are created 5. Robotics used for cDNA amplification and PCR purification 6. Products put onto an array source plate (384 well plate) 7. Printing (using robotics) 8. Microarray created

What are the calculations of normalization factors?

1. Total fluorescence intensity 2. Linear regression analysis 3. Housekeeping genes

What attributes are associated with glass arrays/DNA microarrays?

1. Use glass slides as a matrix (positive charges) 2. Use fluorescent dye-labeling detection (Cy3/Cy5) 3. Have a larger number and higher density of probes than membrane based-arrays (old) 4. Probes are made from either PCR products (cDNA array) or oligonucleotides (oligo array). Probes can be either spotted or in situ synthesized.

How is software used to process images?

1. align spots 2. robot-spotting files integrated with the image 3. local background calculated 4. Reports of spot intensity data exported

How does indirect target labeling occur?

1. cDNA is created from RNA and reverse transcriptase 2. cDNA express ammonia groups, coupling reactions bind these to added dyes 3. End product is separate cDNA, evenly labeled with different dyes

How does image processing work?

1. slides are scanned using confocal laser scanner, red and green lasers excite Cy5 and Cy3. 2. Data from each fluorescence channel is collected and stored as a TIFF image. 3. Software is used to process the images

How does automatic microarray hybridization work?

1. uses hybridization station 2. minimizes handling of microarrays 3. maximizes hybridization through gentle and consistent agitation

What is background subtraction used for?

Allows for comparison of specific signals

What is the concept of microarray technology?

Allows for detection of thousands of genes simultaneously (functional genomics).

What does RNA sequencing allow for?

Analysis of mapped reads allows for estimation of transcript abundance, reads kilobase per million mapped read

What assumption is there for data normalization in microarrays?

For the entire collection of arrayed genes for some subset, the ratio of expression averaged over the set is one

What are microarrays/chips?

Miniaturized arrays having up to tens of thousands of cDNA/oligo (genes) attached to it.

What is microarray hybridization?

The reverse of conventional hybridization (target vs. probe concept).

What is data normalization used for in microarrays?

adjusts for differences in labeling/detection efficiencies for dyes, and the quality of starting RNA

What is K-means clustering used for in microarray data analysis?

divides genes into groups based on their expression patterns, genes with similar expression patterns might be functionally related via the same pathway

What are microarray assays?

hybridization of single-stranded DNA labeled with a fluorescent dye to a complementary DNA attached to the array

How does manual microarray hybridization work?

uses hybridization cassette and cover slips

What are scatter plots used for in microarray data analysis?

view is used to examine the expression levels of genes in two distinct conditions or samples