Microarrays Flashcards
What is a microarray?
- An ordered assembly of nucleic acids immobilised on a solid support
- Usually glass support used
- Usually single stranded DNA that’s immobilised
What is transcriptomics and what is the transcriptome?
- Transcriptomics is the study of the transcriptome
- Transcriptome is the complete set of RNA produced by the genome of an organism
What is transcriptomics used to look at in terms of gene expression?
- looks at the level of gene expression of all the genes expressed within a particular sample
- Also looks at which genes are expressed at different levels between two different types of samples
What type of microarray is used in transcriptomics?
Gene expression microarrays
What are some of the charactersistics of a gene expression microarray?
- There are lots of copies of the same DNA probe in a single spot
- Each spot gives the relative expression for one gene
- Detects the expression of all known genes within a sample because there are so many copies of each probe
What are the 2 methods that are used to produce a gene expression microarray?
- Two colour array (Main technique used)
- One colour array
Briefly describe the two colour array method used to produce a gene expression microarray?
- The RNA from two biological samples (the test sample and control sample) are extracted and then converted into cDNA using reverse transcriptase
- During reverse transcription the cDNA from each biological sample is labelled with a different fluorescent dye, usually Cyanine 3 (Cy3) and Cyanine 5 (Cy5).
- Equal amounts of labelled cDNA are then simultaneously hybridised to the same microarray chip.
- The microarray chip is scanned and each spot on the array represents the relative expression of a particular gene for the test sample and control sample.
Briefly describe the one colour array method used to produce a gene expression microarray?
- RNA from the two biological samples extracted and converted into cDNA via reverse transcriptase
- The cDNA from both biological samples is labelled with the same dye
- The labelled cDNA is then hybridised onto separate microarray chips, one chip for test smaple cDNA and another for the control smaple cDNA.
- The Microarray chips are scanned and the expression levels of a particular gene within each sample is measured using the 2 different chips.
When scanning the fluorescence of each spot on the gene expression microarray chip produced using the 2 colour array method, what colours can be detected and what do each of these colours mean?
- Red - That particular gene is expressed to a higher degree within the test sample compared to the control sample
- Green - That particular gene is expressed to a higher degree within the control smaple compared to the test sample.
- Yellow - That particular gene is expressed to a similar degree in both the control sample and the test sample.
Hierarchical clustering can be used to analyse the results produced from a gene expression microarray. What is hierarchical clustering?
A method of analysis that organises data with similar patterns into classes (clusters).
How can hierachical clustering be used to analyse the results from a gene expression microarray?
- It can be used to cluster the expression of a set of genes into groups based on a particular measurable trait, e.g. body mass index
- So for example you can get an idea of which genes are expressed at a higher or lower rate depending on changes to that measurable trait.
In hierarchical clustering what does the distance between the genes within particular clusters represent?
- Distance between the different clusters represents how different the genes within those different clusters are.
- Clusters of genes that are far away from each other means the genes that make up those clusters are very different form one another
- Genes that are very close together are very similar to one another and so may also be regulated together
Apart from hierarchical clustering what is another way in which gene expression microarray data can be analysed?
Can be analysed using a dendogram
What does distance represent on a dendogram used to analyse gene expression?
Distance represents how similar or different the level of expression for each gene is
In the context of gene expression microarrays what is Quantitative PCR (qPCR) used for?
qPCR is used to confirm the results from a gene expression microarray experiment
When performing qPCR to confirm the results of a gene expression microarray why do you have to convert the mRNA from a test sample into DNA?
It’s because there are no known polymerases that are able to amplify the length of RNA instead of DNA
What is cDNA?
cDNA (complementary DNA) - DNA that is complementary to the original RNA sequence.
When performing qPCR to confirm the results of a gene expression microarray what reason other than the fact that a particular gene is expressed to different levels in different tissues could you see differential expresssion of a gene between tissues?
- Could be due to the fact we started the PCR reaction with different amounts of RNA from different tissues e.g. Lots of RNA from brain, but only a small amount of RNA from the kidney sample.
Why do you run a housekeeping gene as well as a gene of interest when performing gel electrophoresis on the qPCR products?
- Housekeeping genes are expressed at a similar level in all tissues so you’d expect to see a nice strong band in all samples on the gel which would suggest that we started with the same amount of RNA in all the samples
- You can then compare the strength of the band for the housekeeping gene to the gene of interest for the different tissues
- Any difference between the 2 would then be due to experimental variation in the amount of RNA loaded and can be corrected for
This process is called normalisation
In qPCR because you add fluorescent molecules to the reaction mixture there will always be a level of background fluorescence. What is the no. of copies that need to be produced during to produce fluorescence at a greater level than the background fluorescence?
225 copies of the PCR fragment
Define the term “cycle threshold” (Ct)
The cycle threshold is defined as the number of cycles required for the fluorescent signal to cross the threshold (background fluorescence).
What is meant when it is said that PCR can be made into a quantitative technique (qPCR)?
- It means that you modify the technique of PCR so that you are able to use it to count the number of copies of amplified DNA present.
What are the 2 methods that can be used to make PCR quantitative?
- Include a dye in the PCR reaction mix that fluoresces when it binds to double-stranded DNA
- Include a labelled probe in the PCR reaction mixture that only fluoresces when it is incorporated in the PCR product
Explain how qPCR is able to be used to confirm the result of a gene expression microarray?
- Perform qPCR on cDNA produced from RNA extracted from the same biological samples used for the gene expression microarray
- qPCR machine able to measure fluorescence signal as it amplifies cDNA fragments
- Using data from qPCR machine you can plot graph of fluoresence signal compared with the cycle number for the cDNA of each of the samples used
- You then plot the Ct threshold on the graph and use it to find out the Ct value for each of the samples (no. of cycles it takes for fluorescence signal for each sample to go above background fluorescence).
- Once you know the Ct values for each of the samples you are then able to work out which of the samples contained the highest amount of RNA (cDNA)
- Sample with the highest amount of RNA (cDNA) indicates that the genes in that sample were expressed to a higher degree than those same genes within the other samples used.
