Microarrays Flashcards
What are microarrays?
Silicon or glass sheet with thousands of DNA probes that can be used to identify which genes in a tissue are expressed
*the signal from each spot is used to estimate the expression level of a gene
Describe the structure of the 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
Give some Different approaches to measuring mRNA expression.
Two colour Array
One colour Array
Describe the process of the 2-colour microarray.
1) test sample mRNA labelled in red (with Cy5) and converted to cDNA by reverse transcriptase
2) control sample mRNA labelled in green (with Cy3) 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
Describe the process of the 1-colour microarray..
this involves the hybridisation of a single sample (e.g. test/control) to each microarray after it has been labelled
What is done after the 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
What is heirachical 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
What are 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
What is MLAME?
Minimum information about a microarray experiment:
-guidelines that should be included when describing a microarray study
Give some Data Repositories which support MIAME.
ArrayExpress, EBI (European Bioinformatic Institute)
GEO- Gene Expression Omnibus, NCBI (National Centre for Biotechnology Information, USA)
Describe the Technique used to confirm microarray data.
Quantitative PCR (qPCR)
- reverse transcriptase needed to convert RNA to cDNA because normal RNA is not robust enough , which then undergoes normal PCR
- housekeeping genes also used to normalise mRNA levels of genes of interest before the comparison between different samples by the qPCR
How is qPCR made quantitative?
by counting the number of copies of amplified DNA present by using fluorescent molecules (tags)
-fluorescence detected above baseline (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
qPCR is made quantitative by counting number of copies of amplified DNA present by either:
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
What is the use of qPCR in microarray analysis?
used to independently confirm differences in RNA levels between samples
