13- Microarray Flashcards
what is a microarray?
an ordered assembly of nucleic acids immobilised on a solid support (glass side)
used to measure the relative concentration of nucleic acid sequences in a mixture
how are microarrays designed?
microarray is a solid support/ glass slide with each spot containing thousands of identical immobilised probes designed for specific nucleic acid sequences
what is a probe?
short single-stranded oligonucleotide - designed to be complementary to specific target DNA/ RNA sequences
what is the advantage of each spot on the array having thousands of identical probe sequences?
increases the chances of capturing the target sequences during hybridization
describe how microarrays work
thousands of immobilised probes with identical sequences at each spot are immobilised on a solid support
fluorescently tagged genomic DNA samples containing a mixture of different nucleic acid sequences are loaded onto the microarray - complementary DNA seqs. hybridise with complementary probe seqs.
microarray then washed to remove any unbound or non-specifically bound DNA = ensures only specific hybridisations remain
hybridised DNA samples have a fluorescent tag which shows under a laser light
describe the importance of microarrays
looking at gene expression and transcriptomics in tissue - can compare between healthy and unhealthy tissue, allows us to discover the biology of our samples and classify them
SNP genotyping
structural variant detection using array CGH
define a transcriptome
all the genes expressed at a point in time in a tissue = all RNA is the transcriptome
Describe how microarrays are used to measure gene expression levels across the whole transcriptome
two-colour microarray used to detect the expression of thousands of genes at the same time
two samples are used - test and control
mRNA isolated form both samples = represents the transcribed genes
mRNA used as a template, reverse transcribed into cDNA by reverse transcriptase activity
control sample cDNA labelled with one colour, test sample cDNA labelled with another
labelled cDNA from both samples applied to microarray and hybridised with their complementary probes, immobilised on a solid support
the degree of hybridisation is proportional to the level of gene expression in the original samples - cDNA with higher gene expression levels will show more binding
microarray is scanned with a fluorescence scanner which measures intensity - higher fluorescent intensity = more hybridisation = more gene expression in a sample
data is analysed, different colours are assigned to different expression levels:
yellow - equal expression in test and control samples
green - stronger expression in the control
red - expression in the test
red: green fluorescence ratio indicates relative gene expression levels in the two samples
describe the data analysis workflow for microarray gene expression analysis
feature extraction - obtaining raw array data/ cell file
quality control - address anomalies and low-quality spots
normalisation - make data look comparable to other arrays
differential expression analysis - look for (statistically significant) differences between test and control sample gene expression levels, employ t-tests or ANOVA test if needed
biological interpretation - hierarchical clustering to group genes with similar expression classes, network analysis to understand biological significance of identified gene
what is hierarchical clustering? why is it useful?
it’s a method for grouping genes based on their expression patterns - develops gene expression profiles - and expressed in a dendrogram
helps emphasise gene expression differences between classes, between healthy/unhealthy tissue
can be used for disease diagnosis, prognosis, identifying subtypes of tumours and treatment decisions
Describe how microarray results are validated using quantitative PCR (qPCR)
RNA is isolated from the test and control samples and reverse transcribed into cDNA
cDNA used as a template for PCR amplification - house-keeping genes as controls and the gene of interest is amplified
PCR products are run on a gel, visualised as bands
house-keeping genes are controls as they’re expressed at similar levels across tissues = expected to produce bands of similar intensity
unequal bands suggest under-expressed RNA
this method can be made quantitative by fluorescently tagging the copies of amplified DNA - fluorescence will increases proportional to the exponential doubling of copies per cycle
once fluorescence exceeds a set arbitrary level (background fluorescence), the cycle number can be recorded as the cycle threshold. starting out with more DNA/RNA means a lower cycle threshold
method can also be made quantitative by counting the number of copies of amplified DNA - using intercalating dyes (SYBR Green) or labelled probes (TaqMan).
intercalating dyes added to PCR reaction mix, fluoresce when binding to ds DNA. greater fluorescence = proportional to number of copies of amplified DNA
labelled probe in PCR fluoresces when incorporated into PCR product
what is qPCR?
technique used for amplifying and quantifying DNA by measuring the amount of a specific DNA target in real-time
why use qPCR for validating microarray results?
can independently confirm differences in RNA levels between samples
microarray results may have noise, and differences detected may not always be real
what is the clinical application of qPCR validating microarray results?
tumour profiling for breast cancer - e.g. EndoPredict examines 12 different genes to differentiate between various breast cancer profiles and predict recurrence risk
describe how microarrays are used for SNP genotyping and GWAS
GWAS studies used to genotype many SNPs in many subjects - can identify genetic variants associated with disease/ traits
SNP microarrays genotype known SNPs
- in each microarray spot there are 1000s of probes specifically designed to genotype a SNP
- complementary hybridisation between probes and regions of genomic DNA before the SNP of interest
- probe is extended by one fluorescently labelled ddNTP base which is complementary to the base at the SNP position
- 2 different probes designed for each SNP variant to target the two different SNP alleles. probe extension with the fluorescent ddNTP occurs separately or each allele.
- different colour fluorescent tags are used to label the extended probe depending on the switch - allows the microarray to differentiate between a purine to pyrimidine (or vice versa) switch
- interpreting colour signals - a combined signal suggests heterozygous SNP genotypes, a single colour signal suggests homozygous
- software analysis of known SNP - software has determined the possible colours and what they mean in advance
