12. MICROARRAYS Flashcards
How does a microarray work?
- Tagged DNA is added to the microarrays
- Hybridisation will occur between the complementary microarray oligonucleotides & DNA
- Shining a laser will excite the tagged DNA molecules, causing them to glow but this doesn’t happen with DNA that isn’t hybridised
What is a microarray?
- a microarray is an ordered assembly of nucleic acid probes which are immobilised on a solid surface such as silica or glass
Give three uses of microarrays
- Transcriptomics - gene expression
- SNP arrays - SNP genotyping
- Array CGH - structural variants such as CNV
What is transcriptomics?
- TRANSCRIPTOMICS is the study of the transcriptome & it’s functions
- The transcriptome is the all the mRNA produced from the DNA in an organism
What’s the difference between one colour & two colour arrays?
- One colour arrays - measures the gene expression from each sample on SEPARATE arrays
- Two colour array - compares the relative the gene expression from samples on a SINGLE
array. The two samples are labelled with different fluorescent dyes (Cy3 & Cy4) which can then be measured after hybridisation
What are the data analysis workflow steps of microarrays?
- Feature extraction
- Quality control
- Normalisation
- Differential expression analysis
- Biological interpretation
What are the 4 steps in between normalization & biological interpretation?
Normalisation –> Hierachial clustering –> Gene filtering –> Statistical test –> Generate gene list –> Biogical interpretation
What is clustering in the context of microarrays?
- Clustering is a way of organising data with similar patterns into classes
- The ‘objects’ within a class are more related than the objects outside a class
What type of diagram shows clustering in a microarray?
- Clustering can be shown as a DENDROGRAM
- A dendrogram is a tree, it’s an alternative way of showing similarity between samples
What does qPCR do with microarray results?
- qPCR is a way to confirm microarrays
- Amplifying the RNA can allow us to see how much RNA was present in the sample at the beginning
- Initially RNA is converted to cDNA by reverse transcriptase
- Both cDNA & the housekeeping gene undergo PCR
How can qPCR or RT-PCR be made quantitative?
- We can make q-PCR quantitative by counting the number of amplified copies of DNA present
- But, we cannot physically count the copies so we can count the fluorescent molecule that DNA is tagged with
*What are two ways to count the number of amplified molecules from qPCR?
- Include a dye in the PCR reaction mix which shows up as fluorescent when it binds to the double stranded DNA such as SYBR green
- Label a probe in the PCR with a molecules that only fluoresces once it’s been incorporated into the PCR product such as TaqMan
*What is the Ct value & what does it show?
- The CT value is the Cycle threshold number. It is defined as the number of cycles where fluorescence exceeds background levels
- In exponential PCR, the CT is 225
- The higher the starting amount of cDNA/RNA, the lower the Ct value
Why is qPCR chosen to confirm microarrays over other techniques?
- Other techniques have disadvantages
- Probe binding is noisy & doesn’t find a real difference, if it does it tends to be small
- RNA sequencing is more accurate & more reproducible but it’s more expensive
What are SNP microarrays?
- Microarrays hybridise with genomic DNA adjacent to the SNPs
- The SNPs are then extended by a single bases which is fluorescently labelled & detected witha high detection scanner
