microarrays Flashcards
Microarray
Silicon or glass sheet with thousands of ssDNA probes that can be used to identify which genes in a tissue are expressed
asses gene of interest AND housekeeping gene as a reference
*the signal from each spot is used to estimate the expression level of a gene
Probes in a microarray
single stranded oligonucleotides immobilised on the array so that DNA or RNA can hybidise to them
How is probe-target hybridisation quantified?
by detection of fluorescently labelled targets to determine the relative abundance of nucleic acid sequences in the target
Different approaches to measuring mRNA expression
Two colour Array
One colour Array
Two-Colour Microarray
1) test sample mRNA labelled in red and converted to cDNA by reverse transcriptase
2) control sample (housekeeping gene) mRNA labelled in green and converted to cDNA by reverse transcriptase
3) mix together and hybridise them onto single array
4) if only expressed in a test sample, signal will be red; if only expressed in control sample, signal will be green; if expressed in both, signal will be yellow
One Colour Microarray
this involves the hybridisation of a single sample (e.g. test/control) to each microarray after it has been labelled
After microarray is produced:
1) feature extraction where the data is taken from the array and creating a file that contains all of the array information on it
2) quality control
3) normalisation
4) differential expression analysis
5) biological interpretation
6) submit data to a public repository
Heirarchical Clustering
most popular method for gene expression data analysis:
- gene with similar expression patterns are grouped into classes and are connected by a series of branches (clustering tree or dendrogram)
- objects within a class are more similar to each other than to objects outside the class
Data Repositories
as microarray experiments are expensive, microarray data repositories, which contain microarray gene expression data, are used so people can share their data for further analysis and interpretation
MIAME
Minimum information about a microarray experiment:
- guidelines that should be included when describing a microarray study
Technique used to confirm microarray data
Quantitative PCR (qPCR)
How is qPCR made quantitative?
by counting the number of copies of amplified DNA present by using fluorescent molecules (tags) - basically count the fluorescent tags
- fluorescence detected above baseline of 225 copies (and therefore Ct number) is statistically significant because this threshold must be established to quantify the amount of DNA in the samples
The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (ie exceeds baseline level).
*the higher the amount of starting RNA (cDNA), the lower the Ct value, meaning the quicker that fluorescent threshold is reached
how do you count the number of amplified fluorescent tags in qPCR?
including a dye in the PCR reaction mix that fluoresces when it binds double-stranded DNA e.g. an intercalating dye such as SYBR Green
OR
labelling a probe in the PCR that only fluoresces when it is incorporated in the PCR product e.g. TaqMan
qPCR in microarray analysis
used to independently confirm differences in RNA levels between samples
what is a more accurate measure of RNA transcript analysis than qPCR
RNA Sequencing (Next Gen Sequencing) because it is more reproducible and works over a much wider range of concentrations
- disadvantage: more expensive
