Microarrays

Question 1

What is a microarray?

Answer 1

• An ordered assembly of nucleic acids immobilised on a solid support
• Usually glass support used
• Usually single stranded DNA that’s immobilised

Question 2

What is transcriptomics and what is the transcriptome?

Answer 2

• Transcriptomics is the study of the transcriptome
• Transcriptome is the complete set of RNA produced by the genome of an organism

Question 3

What is transcriptomics used to look at in terms of gene expression?

Answer 3

• 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

Question 4

What type of microarray is used in transcriptomics?

Answer 4

Gene expression microarrays

Question 5

What are some of the charactersistics of a gene expression microarray?

Answer 5

• 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

Question 6

What are the 2 methods that are used to produce a gene expression microarray?

Answer 6

• Two colour array (Main technique used)
• One colour array

Question 7

Briefly describe the two colour array method used to produce a gene expression microarray?

Answer 7

• 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.

Question 8

Briefly describe the one colour array method used to produce a gene expression microarray?

Answer 8

• 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.

Question 9

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?

Answer 9

• 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.

Question 10

Hierarchical clustering can be used to analyse the results produced from a gene expression microarray. What is hierarchical clustering?

Answer 10

A method of analysis that organises data with similar patterns into classes (clusters).

Question 11

How can hierachical clustering be used to analyse the results from a gene expression microarray?

Answer 11

• 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.

Question 12

In hierarchical clustering what does the distance between the genes within particular clusters represent?

Answer 12

• 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

Question 13

Apart from hierarchical clustering what is another way in which gene expression microarray data can be analysed?

Answer 13

Can be analysed using a dendogram

Question 14

What does distance represent on a dendogram used to analyse gene expression?

Answer 14

Distance represents how similar or different the level of expression for each gene is

Question 15

In the context of gene expression microarrays what is Quantitative PCR (qPCR) used for?

Answer 15

qPCR is used to confirm the results from a gene expression microarray experiment

Question 16

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?

Answer 16

It’s because there are no known polymerases that are able to amplify the length of RNA instead of DNA

Question 17

What is cDNA?

Answer 17

cDNA (complementary DNA) - DNA that is complementary to the original RNA sequence.

Question 18

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?

Answer 18

• 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.

Question 19

Why do you run a housekeeping gene as well as a gene of interest when performing gel electrophoresis on the qPCR products?

Answer 19

• 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

Question 20

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?

Answer 20

225 copies of the PCR fragment

Question 21

Define the term “cycle threshold” (C_t)

Answer 21

The cycle threshold is defined as the number of cycles required for the fluorescent signal to cross the threshold (background fluorescence).

Question 22

What is meant when it is said that PCR can be made into a quantitative technique (qPCR)?

Answer 22

• 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.

Question 23

What are the 2 methods that can be used to make PCR quantitative?

Answer 23

• 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

Question 24

Explain how qPCR is able to be used to confirm the result of a gene expression microarray?

Answer 24

• 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 C_t threshold on the graph and use it to find out the C_t 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 C_t 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.

Question 25

What problem is there with gene expression microarrrays that means that qPCR needs to be used to confirm the results of a gene expression microarray?

Answer 25

* Probe binding to an array is noisy and differences in gene expression between samples can be detected that are not real, especially where differences are small

Question 26

Why can you sometimes get false differences in gene expression between samples when performing a gene expression microarray?

Answer 26

Because cDNA from the samples can hybridise to probes that are not completely complementary.

Question 27

Why is RNA-seq a better technique for assessing differences in gene expression for genes within a particular sample compared to a gene expression microarray?

Answer 27

* RNA-Seq is a more accurate measure of RNA transcript abundance * It's more reproducible * Works over a much wider range of concentrations of the original RNA

Question 28

Give an example of a therapeutic use of qPCR

Answer 28

* In the NHS qPCR is used as part of tumor profiling tests which guide decisions on whether or not to use chemotherapy in early breast cancer * Results from the qPCR are used to give predictions about the risk of breast cancer recurrence over the next 10 years * This recurrence risk guides decision on whether or not to use chemotherapy

Question 29

What are some of the characteristics of a SNP microarray?

Answer 29

* Each spot contains lots of copies of the same oligonucleotide probe. * Each spot is used to detect a particular SNP

Question 30

How does a SNP microarray work?

Answer 30

1. You take lots of copies of a particular probe and attach each copy to a different spot on the glass slide. 2. You do the same thing for every single SNP that you’re interested in 3. We then take fragmented genomic DNA from our patient and wash it over the slide 4. The genomic DNA will then hybridise to its complementary probe 5. You then extend the immobilised oligonucleotide probe by one base using dideoxy nucleotide triphosphates (ddNTPs) with a fluorescent tag 6. These tagged ddNTPs will hybridise with the base on the fragmented genomic DNA where the SNP occurs 7. The fluorescence the ddNTPs give off is then used to see whether a person is homozygous or heterozygous for that particular SNP or whether they have it at all

Question 31

When scanning the fluorescence of each spot on the SNP microarray chip, what colours can be detected and what do each of these colours mean?

Answer 31

* Red - Person is homozygous for a particular SNP as they have DNA sequences that only contain one of the 2 possible bases at the psoition where the SNP is * Green - Person is homozygous for a particular SNP as they have DNA sequences that only have the other of the 2 possible bases where the SNP is * Yellow - Person is heterozygous for a particular SNP as they have DNA sequences which contain both of the 2 possible bases at the position where the SNP is

Question 32

When performing a SNP microarray why do different DNA fragments hybridise to the same probe at different points?

Answer 32

* The fragmentation of the genomic DNA is random so you end with different lengths of DNA * This means that you end up with different parts of the genomic DNA being complementary to the probe so each fragment binds to the probe at a different point

Question 33

What are SNP microarrays used for?

Answer 33

They are used in Genome-wide association studies (GWAS) to identify SNPs within the genome of a person that are associated with a particular disease

Question 34

What is Array Comparative Genomic Hybridisation (aCGH) used for?

Answer 34

Array CGH is used to detect structural variants (copy number variants) within a person’s genome.

Question 35

How does Array CGH work?

Answer 35

* The entire genome of a patient is fragmented and the same is done for the control DNA which is taken from a similar source as the patient DNA * The patient DNA and the control DNA are labelled with Cy3, green, and Cy5, red. * They are then mixed together and hybridised to the microarray which contains lots of DNA fragments * The patient and control DNA compete to hybridise with the DNA fragments on the array * Each spot on the array is then scanned to identify the partcular colour fluorescence that it produces

Question 36

When scanning the fluorescence on each of the spots on an Array CGH chip what colours can be detected and what does each colour mean?

Answer 36

* Green - Means too much patient DNA has hybridised to that spot on the array compared to the control DNA - This means there's been a duplication of a copy of that particular gene in the patient DNA * Red - Means too much control DNA has hybridised to that spot on the array compared to the patient DNA - This means there's been a deletion of a copy of that particular gene in the patient DNA * Yellow - Equal amounts of patient DNA and control DNA have hybridised to that spot on the array - This means that the no. of copies of that particular gene are the same as in the control DNA has so copy no. variation hasn't occured to that particular gene