Microarrays

Question 1

what is a microarray

Answer 1

an ordered assembly of nucleic acids immobilised on a solid support

support = glass = like a microscope slide

Question 2

describe microarray probes

Answer 2

probes = short pieces of ssDNA immobilised on surface of the array

• are oligonucleotides
• each spot on array consists of thousands of probes with same sequence

Question 3

what are the expression levels of all genes in my sample

Answer 3

the transcriptome

Question 4

which genes are expressed at different levels between two different types of samples

Answer 4

discover the biology of your samples
classify samples
predict which class a sample belongs to

Question 5

describe gene expression microarrays

Answer 5

lots of copies of same probe in a spot

each spot gives the relative expression for one transcript

detects all known transcripts in one sample

Question 6

describe expression analysis

Answer 6

1. normalisation
2. hierarchical clustering
3. gene filtering
4. statistical tests
5. generate gene list
6. biological interpretation

Question 7

describe clustering

Answer 7

organises data with similar patterns into classes 
objects within a class are more similar to each other than to objects outside the class

Question 8

describe data repositories

Answer 8

maximise utility of microarray = share data and use other peoples data

if user provides minimum info about microarray experiment = easier to compare results

array express

GEO = gene expression omnibus

Question 9

q-pcr

Answer 9

we can make rt-pcr quantitative by counting number of copies of amplified DNA present

we count the copies by using fluorescent molecules = taks

Question 10

how do you count the number of amplified molecules present

Answer 10

• include a dye in the PCR reaction mix that fluoresces when it binds double-stranded DNA
• label a probe in per that only fluoresces when it is incorporated in the PCR product

Question 11

why qPCR

Answer 11

• used to independently confirm differences in RNA levels between samples
• probe binding is noisy and differences can be detected that are not real, especially where differences are small
• RNA seq is a more accurate measure of RNA transcript abundance, it is more reproducible and works over a much wider range of concentrations, but more expensive

Question 12

breast cancer endopredict risk estimation

Answer 12

• at low score, endocrine therapy alone is sufficient
• at higher scores, et+c os beneficial compared to ET alone

this test ensures that only patients who will benefit from chemotherapy will receive it

Question 13

SNP microarrays

Answer 13

genome wide association studies are only possible as we can genotype large numbers of SNPs in large numbers of subjects

this is possible by using microarrays that hybridise with genomic DNA adjacent to SNPs

the SNP is extended by one base that is fluorescently labelled and detected using high definition scanner

Question 14

describe a spot

Answer 14

lots of copies of same single stranded oligonucleotide probe

each probe is for genotyping one SNP

Question 15

describe genotyping microarrays

Answer 15

each spot gives genotype for one SNP

up to 5 million spots per sample on array

genome-wide analysis possible

Question 16

SNP genotypes

Answer 16

software translates the 3 diff colour signals for each probe into genotypes

a few SNPs are reviewed by hand but most are not