L15: Microarrays Flashcards
What is a microarray?
A substrate of some kind (e.g. a glass slide) on which we have thousands of copies of the same short stretches of DNA, named probes
What are ‘probes’?
The same short stretches of DNA
How are probes used in microarrays?
Probes are complimentary in sequence to things in sample, which we will have extracted the nucleic acid from that we want to interrogate
What does the term ‘features’ to?
Features are spots where probes are arranged, which have thousands of copies of the same probe’s sequence (in that feature)
What mechanism do microarrays employ?
They work by hybridisation (watson-crick base pairing between probe and sample)
How do microarrays use hybridisation?
- DNA/ RNA sample we want to bind is called the Target
- Results are then read out by target being labelled with a fluorescent marker
- Fluorescence intensity of feature relates to amount of target DNA in sample
- Allows us to observe changes for specific genes
Name the 3 main applications of microarrays
Gene expression, SNP arrays/ affymetrix SNP6 arrays & methylation arrays
How were microarrays were used for gene expression?
- arrays run under different conditions, allowing for differential gene expression as reported fold-changes
What does use of microarrays in investigating gene expression allow?
Allows us to see what genes are switched on by cell response in different conditions; these can be investigated or characterisation
Explain the use of SNP arrays/ affymetrix SNP6 arrays?
Probes are designed for different alleles, ratio of intensity allows us to see if SNP is reference or alternative allele
- copy # of each SNP can also be inferred via computer, allowing detection of chromosomal (large-scale) or focal duplications/ losses
How are SNP arrays/ affymetrix SNP6 arrays useful?
They allow for GWAS, where gene linkage is found between SNP markers and phenotypes
- requires large patient cohorts
What does ‘SNP’ stand for?
Single nucleotide polymorphism (change)
How are methylation arrays carried out?
Via illumina 450K or EPIC methylation arrays
How are methylation arrays useful?
Allows the methylation state of DNA to be interrogated
- important in epigenetic regulation of chromatin structure and in cancer
- cytosine bases that occur before a guanine can be methylated
What can methylation of promoter regions of genes lead to?
The gene being silenced while being demethylated causing the over-expression of genes
What does bisulfate treatment cause?
The conversion of cytosine to uracil and then thymine but methylation of cytosine at CpG sites protects bases from this conversion
- probes exist for each C or T state of a CpG
- ratio intensity here (beta-value) allows us to detect methylation state of CpG
Explain the process of Northern Blotting
- Sample of tissue mashed up then RNA extracted using nucleic kit
- Then run on agarose gel (electrophoresis) with formaldehyde and seperated by size
- Blot RNA onto nylon membrane since RNA probes can’t enter gel, creating complimentary bits of RNA identical to gene sequences interested in quantifying
- Probes labeleed with fluorescent P and hybridise into this membrane and base pair against genes of interst
Nylon membrane then put through beta decay and on x-ray film and placed in freezer or via phosphoimager freezer then developed
Explain the process of microarrays
- RNA extracted from sample and converted using reverse transcriptase to cDNA as mRNA isn’t stable. cDNA lablled w fluorescent dye
- cDNA sample hybridised into microarray. Array has ssDNA probes in features and the more mRNA, the more fluorescence visible
- Data is processed on computer and we end up with a fold change, due to comparing control to treated sample
Why are microarrays now used over northern blotting? (nb)
- Microarrays are less time consuming and laborious as it’s an automated process
- Not radioactive, unlike nb
- Microarrays can work on 10,000 of genes, while nb can only work on a handful
- Microarrays provide much higher dynamic range but nbs provides mainly qualitative data
- Microarrays don’t need hypothesis, unlike nb, in advance to decide which genes wanted to be investigated
How do spotted cDNA microarrays work?
Spot cDNA probes onto microscopic slides and later robots used to spot slides
- common to run 2 samples on the same arrray using green Cy3 and red Cy5, as one channel would be control
What have spotted cDNA microarrays been replaced with?
Commerically produced arrays, which use oglionucleotide prones synthesised on a chip in place of bound cDNAs on glass slide
Why are commercially produced arrays preferred over spotted cDNA microarrays?
- commercial arrays have probes synthesised in situ on chip
- results are more comparable due to standard experimenal procedures, automation for sample prep and hybridisation and washing
- no complicated dye swapping experimental design
- uses cRNA targets labelled with single fluorochrome
- a single sample is hybridisd to a single chip
Name limitations of microarrays
It’s not easy to compare them due to
- difference in sample extraction yield (mRNA)
- differences in hybridisation conditions
- differences from one array to the next and possible artefacts on array
How is quality control ensured on microarrays?
- Have 3 of each array, minimum
- Batch effect: show up where sample variability is due to sampling handle, rather than biological variation e.g. were samples extracted/ hybridised on the same day or by the same person
