Transcriptomics. Flashcards
Define a conctamer?
A strand of cDNA that is formed when multiple cDNA fragments are linked together.
Define a microarray?
A technique that is used to look at gene expression.
Define a macroarray?
A larger version of a microarray.
Define a snapshot?
When a scientific procedure gives results that are only relevant for a specific time.
E.g. Transcriptomics.
Define SNP chips?
Commonly used to detect point mutations.
Define a tiling array?
A technique that is used to look at gene expression across a genome or chromosome.
What is transcriptomics?
The process of analysing all of the mRNA within a cell at a given time.
How does transcriptomics analyse DNA?
It analyses the percentage of the genetic code that is transcribed into mRNA.
Why is transcriptomics context dependent?
As the amount of mRNA within a cell depends on when that cell is analysed.
Why is genomics not context dependent?
Because the number of genes in the organism never changes.
What 4 things does the transcription of DNA into mRNA depend on?
Environment.
Development.
Time of day.
Particular tissue that is being analysed.
What is the major difference between transcriptomics and genomics?
Transcriptomics gives scientists an idea about how genes are expressed within a certain cell or tissue.
Genomics gives an analysis of gene expression throughout the entire body.
What does the study of transcriptomics allow us to compare?
To make compare gene expression in different tissues e.g;
What are 4 comparisons that we can make via the use of transcriptomics?
Different tissues within the same organism e.g. liver cell Vs brain cell.
Same tissues within the same organism e.g. tumour cell Vs non tumour cell.
Same tissues within different organisms e.g. wild type cells Vs mutant cells.
How gene expression changes in certain tissues during development.
What biomolecules will genomics and transcriptomics both study?
The structure and chemical composition of the nucleic acids.
What 3 analytic processes will genomics and transcriptomics use to ananlyse nucleic acids?
Electrophoresis.
Hybridisation.
Sequencing.
What is the biggest challenge of studying RNA strands?
mRNA is very fragile.
How do scientists make mRNA less fragile?
They will use reverse transcriptase to convert coding mRNA back to DNA.
What is one of the major benefits of transcriptomics when referring to mRNA?
It can be used to identify particular classes of mRNA and determine their abundance within the cell.
What are 4 of the major benefits of transcriptomics when referring to specific genes?
It suggests which genes are involved in which biological processes.
It allows scientists to group genes with similar functions together.
It tells scientists which genes are expressed under particular environmental conditions.
It can help to identify genetic markers for disease and to identify changes within the proteome.
What molecular technique is usually used in conjunction with the hybridisation of mRNA?
Northern blot.
What needs to be present for hybridisation processes involving mRNA?
An oligonucleotide containing a sequence of nucleotides identical to a sequence of nucleotides on the cDNA.
What is cDNA formed from?
From the protein coding regions (exons) of mRNA.
How is cDNA made from mRNA?
Via reverse transcription.
What happens during the hybridisation of mRNA after the cDNA has been formed?
The cDNA is spliced by restriction enzymes and will then undergo electrophoresis.
What happens during the hybdrisation of mRNA after the cDNA has been cut into fragments?
The oligonucleotide probe is added.
What happens during the hybridisation of mRNA after the probe has been added to the cDNA fragments?
If the probe hybridises to the cDNA then a fluorescent signal will become visible.
This will tell us that the specific sequence of nucleotides was present within the cDNA.
Why does transcriptomics often utilise the process of PCR?
To amplify sections of coding DNA.
What will scientists do with a piece of cDNA after it has undergone PCR?
They can use individual copies for many different modes of analysis.
What does the reporter gene technique of transcriptomics involve?
It involves the fusion of a gene of interest with a reporter gene which code for certain proteins.
What are 2 common reporter genes that used in transcroptomics?
Luciferase or glucouronidase.
How does the reporter gene technique work?
If any changes occur in the gene of interest then the reporter gene is expressed and acts as a warning.
What industry are reporter genes commonly used in?
In crop farming where the reporter gene will be expressed if the crop has been damaged by insects.
What happens during the hybridisation of mRNA after the cDNA has been formed during the detection of transcriptional changes within a single gene?
The cDNA is spliced by restriction enzymes and will then undergo electrophoresis.
What is the difference between viewing results in a microarray or a macroarray?
Hybridisation results can be clearly seen in a macroarray, whereas they cannot be visualised in a microarray.
What do macroarrays allow for?
For the expression levels of thousands of genes to be detected in a single experiment.
What are the 2 most common sequencing based techniques used when detecting transcriptional changes within multiple genes?
Serial analysis of gene expression (SAGE).
Massively parallel signature sequencing (MPSS).
Do the sequencing based techniques used for detecting transcriptional changes within multiple genes require any knowledge of the order of nucleotides within the cDNA molecule?
No.
Do the PCR and hybridisation based techniques used in for detecting transcriptional changes within multiple genes require any knowledge of the order of nucleotides within the cDNA molecule?
Yes, as PCR requires the presence of forward and reverse primers.
Hybridisation requires the presence of complimentary probes.
What do MPSS and SAGE and allow scientists to detemrine?
The exact order of nucleotides so that they can can build primers or probes.
Does MPSS and SAGE require knowledege of the coding sequence?
No.
So this technique serves as a universal platform to study any mRNA transcript.
What is the first step of perfroming SAGE analysis?
Removing mRNA from a tissue sample e.g. a tumour or a specific tissue within an organism.
What happens in SAGE after the tissue has been extracted?
It is converted to cDNA.
Restriction enzymes will extract small sequences of DNA from pre-determined positions within the cDNA molecule.
What happens to the small fragments of DNA that have been formed by restirciton enzymes during SAGE?
They are linked together to form a conctamer which can then be sequenced by traditional sequencing techniques.
How long are the sequence fragments that are created during SAGE?
Between 9 and 14 base pairs.
What can the sequence fragments used in SAGE tell scientists?
Which genes are used to code for a particular piece of RNA.
What is the first step of MPSS after the cDNA fragments have been formed?
The small molecules of cDNA are cloned.
What happens in MPSS after the cDNA fragments have been formed?
They are attached to a fluorescent primer and a tag.
What kind of fluoresent primers are the cDNA fragments attached to in MPSS?
The fluorescent primer will differ for each different class of cDNA.
E.g. Different primers are added to cDNA from different tissues.
What is the tag that is attached to the cDNA in MPSS made from?
Sequences of known DNA.
What happens to the cDNA copies after they have been attached to their fluoresent markers and tags?
They are attached onto microbeads.
How are the microbeads that are used in MPSS made?
Through the use of LYNX megaclone technology.
What is attached to the microbeads that are attached to the cDNA copies in MPSS?
ThE microbeads are attached to molecules known as anti-tags.
Why are the microbeads used in MPSS attached to antitags?
They are complimentary to the tags on the cDNA.
This allows complimentary bonds to form between the tags on the cDNA and the anti-tags on the microbeads.
What happens in MPSS after the cDNA copies have been attached to the microbeads?
They are sorted into categories based on the type of fluorescent primer that they contain.
Where are the DNA molecules attached to the probes during MPSS?
At their poly-A end.
What happens in MPSS after the microbead/cDNA molecules have been sorted?
Sequencing can occur.
What are expression arrays consist of?
Of cDNA arrays that contain features such as known oligonucleotides.
How can an expression array be used to to identify sets of genes?
cDNA can be placed into the array to see if hybridisation occurs.
What can expression arrays be used for?
To identify sets of genes.
To identify all the genes that are expressed within a certain cell.
What is a cDNA based microarray based on?
The ability of a given mRNA molecule to hybridise to its original coding sequence in the form of a cDNA template.
What should an mRNA molecule be able to bind to?
To its cDNA template.
What is the first step of performing a microarray to detect gene expression?
To extract diseased tissue and remove the mRNA.
This step is repeated, but the mRNA is removed from healthy tissue.
Both sets of mRNA are then converted to cDNA.
What happens to the 2 sets of cDNA in a microarray after the cDNA has been formed?
The healthy cDNA is labelled with CY-3 which is a green fluorescent dye.
The diseased cDNA is labeled with CY-5 which is a red fluorescent dye.
What happens when performing a microarray after the 2 sets of cDNA have been labelled with fluoresent dyes?
Equal amounts of the labelled cDNA’s are added to a microarray so they can be analysed.
How will the microarray appear if there is more expression from the healthy cDNA?
The array will turn green.
How will the microarray appear if there is more expression from the diseased cDNA?
The array will turn red.
How will the microarray appear if there are equal amounts of expression from the diseased and healthy cDNA?
The array will turn yellow.
How will the microarray appear if no genes are expressed at all?
The microarray will remain blue.
What makes reading the results of microarrays very difficult?
The spots are very small and it requires robots or computers to be able to detect any change in colour.
What kind of microarrays utilise competitive hybridisation?
The microarrays that use of 2 colours.
How does a microarray that uses competitive hybridisation work?
1 particular cDNA will compete with another to hybridise to the probe.
The cDNA that can bind to the probe will dictate the colour of the microarray.
What kind of microarrays utilise non-competitive hybridisation?
They will only use fluorescent indicators of 1 colour.
What is cross species hybridisation?
A microarray that can investigate the similarities in cDNA from different species.
What does cross speices hybridisation involve?
Selecting cDNA from 2 different species and seeing if it will hybridise to a probe.
What is an affymetrix gene fix?
A type of microarray that can be used to compare different strands of cDNA.
What probes are used in an affymetrix gene fix?
Oligonucleotides that are fixed onto specific chips.
How does an affymetrix gene fix work?
The cDNA of interest will hybridise to one of the thousands of probes on the chips.
How many chips are used per DNA sample in the affymetrix gene fix technique?
Each sample of DNA uses a single chip.
How many chips are used per experiment in the affymetrix gene fix technique?
One chip is used per experiment.
How is mRNA prepared to undergo the affymetrix gene fix technique?
The mRNA is converted to cDNA and fluorescent markers are added to it.
What happens in the affymetrix gene fix technique after the cDNA has been formed?
The cDNA is added to the chips and if the cDNA is complimentary to the probes then hybridisation will occur.
How do we know if hybridisation has occurred in the affymetrix gene technique?
The fluorescent markers.
Is the array used for the affymetrix gene competitive or non-competitive?
Non competitive as it uses a single colour and only one sample of genetic material is used per chip.
Are 2 channel microarrays an example of a competitive or non-competitive microarray?
Competitive microarray as it uses cDNA from different sources.
What happens to the cDNA that is used in a 2 channel microarray?
The cDNA strands are labeled with fluorescent probes of different colours.
What happens to the cDNA probes in a 2 channel microarray after the fluoresent probes have been added?
The 2 tissues are added to the microarray and they will compete with each other to hybridise to the probe.
What will show the degree of hybridisation in a 2 channel microarray?
The colours that are expressed.
E.g. red would indicate that one tissue had a higher degree of hybridisation.
Green would indicate that the other tissue had a higher degree of hybridisation.
Orange would mean that both sets of cDNA have equal amounts of hybridisation.
What does MIAME stand for?
Minimal Information About a Microarray Experiment.
What is the function of MIAME?
To dictate the international standard for microarray experiments.
Why does MIAME state that results from a microarray experiment must be published internationally?
So that they can be reproducible.
What should happen if 2 people in different labs carry out the same microarray experiment?
They should get the same results.
What is the function of the MGED is the Microarray Gene Expression Data society?
To store the data from microarray experiments so that it can be accessed by other people.
How can mircoarrays be used to detect certain diseases?
The can detect the gene expression patterns of certain diseases and this can help with the diagnosis of disease.
How can microarrays be used to detect cancer cells?
They can monitor the cell cycle which helps to detect cancerous cells.
How can microarrays be used to detect drug metabolism?
By monitoring changes gene expression once drugs have been used.
Can microarrays be used to detect changes in gene expression?
Yes.
Why would scientists want teo detect changes in gene expression?
To see how an organism is developing.
To detect changes in gene expression between males and females.
To detect changes in gene expression between individuals of different species.
To see how gene expression changes under different environmental conditions.
What are tiling arrays used for?
To evaluate the genome or chromosome of an organism.
What kind of probes are used in tiling arrays?
Multiple high density probes that will each bind to a specific region of the genomic DNA.
How will the high density probes on a tiling array arrange themselves on a chromosome?
They can either overlap each other or lie adjacent to each other.
What does a tiling array allow for the detection of?
Specific genes on a chromosome.
What is the first step of a tiling array?
To form a single strand of all of the cDNA from a chromosome.
What happens in a tiling array after the single strand of cDNA has been formed?
Probes that contain the same sequences as specific genes will be added to the array.
What happens in a tiling array after the probes have been added to the array?
They will hybridise to the cDNA and we can know that that particular chromosome contains that gene.
What do tiling arrays allow us to identify?
Regions of a chromosome that are transcriptionally active.
What do tiling arrays allow us to discover?
New exons that were previously unknown.
Exons that may have modified by mutations, poly-adenylation or alternative splicing.
What methods of transcriptimics are used to detect transcriptional changes within a single gene.
Hybridisation based techniques.
PCR based techniques.
Reporter gene techniques.
What methods of transcriptimics are used to detect transcriptional changes within a multiple genes.
PCR based techniques.
Hybridisation based techniques.
Sequencing based techniques. (SAGE and MPSS).
Expression arrays.
