High throughput (omic) techniques

Question 1

What are the omics?

Answer 1

The omics enable the global analysis of DNA (genes), RNAs and proteins
1000s of genes, RNAs and proteins can be analysed simultaneously
Enables non-hypothesis based approaches to study cells in health and disease
Instead of looking at one gene we can look at all genes simultaneously
Each approach has provided new insights in cell function and disease

DNA - genomics
RNA - transcriptomics
Protein - proteomics

Question 2

Describe genomics in cell biology?

Answer 2

Genomics is the study of an organism’s or a virus’ genome
Sequencing genomes by determining the order of A, G, C and T (U in RNA viruses)
The major advances - development of dideoxy chain termination sequencing (Sanger method)

Question 3

Describe next generation sequencing?

Answer 3

NGS uses massive parallel sequencing to generate millions of short (50-200bp) sequence reads that can be aligned by computational methods to produce whole genome sequences
It builds up the sequence from the strand/primer
HiSeq sequencing systems manufactured by Illumina will sequence 1X human genome in 1 hour
This uses different dyes for the different bases - so we can take a photograph, and cleave off the dye
This sequential photography is repeated to build up the sequence
This used to be very laborious - but not now, including cheaper costs now

Question 4

How are genomics used in cell biology?

Answer 4

Mapping of polymorphisms/genes linked to inherited disease

Mapping of somatic DNA changes linked to disease: cancer

Question 5

Describe cancer genomics?

Answer 5

Cancer is caused by uncontrolled growth of cells and their spread to other sites and is associated with mutations and other changes in the cancer cell genome
Mutations can be inherited, but most occur specifically within the cell
Examining the cancer cell genome provides information about the malignant transformation of cancer cells
You can determine how the cells have become transformed and evolved from the original non-cancerous cells and why they metastasize
This will help develop new targeted treatments for the cancer

Question 6

Give an example of cancer genomics?

Answer 6

Hepatocellular carcinoma (liver cancer) is the 3rd most common cause cancer 
Associated with hepatitis B or C, alcoholism, or non-alcoholic fatty liver disease
The genome changes in exons from HCC were mainly missense mutations (74%), with small insertion and/or deletion (14%) and nonsense and splice-site (12%) modifications

By looking at the roles of the genes that were effected (pathways disregulated by the tumours)
Guichard used this DNA sequence information to identify cellular pathways affected in in HCC
In order of frequency
1. Wnt/β-catenin pathway
2. p53 pathway
3. Chromatin remodelling
4. PI3K/Ras signalling
5. Oxidative stress ad ER stress pathways

Question 7

Give another example: of cancer genomics?

Answer 7

NGS analysis of genomes from single cells
Tumours are typically heterogeneous mixtures of cells, analysing the DNA of an entire tumour mass will not provide a picture of the heterogeneity of the tumour
Navin used single cell sequencing to analyse 2 breast tumours and liver metastasis on 100 cells from each tumour
Single nuclei isolated and their genomes sequenced
One primary tumour was formed from expansion of a single cell with one cell seeding the metastatic tumour with little further evolution,
The other tumour was formed by 3 subpopulations that emerged when the tumour was smaller - metastatic
Shows how they evolve from the original tumour - learning about the development of the cancer

Question 8

Describe metagenomics?

Answer 8

Metagenomics: environmental, community or population genomics
Not the analysis of discreet genome, but analyses a collection of genetic material in a sample i.e. multiple genomes
First developed in studies to examine non-culturable organisms - now has a wide range of applications

Question 9

Give an example of metagenomics?

Answer 9

Metagenomics of gut microbiome
10^14 microorganisms live in or on our bodies i.e. 10X greater than the number of human cells
Most microorganisms reside in the gut where they play important roles, but changes in gut microorganisms may be associated with bowel diseases
There is reduced diversity of gut microorganisms in subjects with inflammatory bowel disease

Question 10

Describe transcriptomics in cell biology?

Answer 10

Transcriptomics is the study of mRNA molecules inside organisms, tissues and cells
With some exceptions (eg mature B and T cells and cancer cells), the genome of all the cells in the body should be identical
By contrast the transcriptome of different cells and tissues is different
The transcriptome represents a point in time and the genes that are being expressed and the level they are expressed
This will change over time due to the effect of many factors

Question 11

How do we analyse the transcriptome?

Answer 11

High throughput analysis of the transcriptome uses either - DNA microarrays or more commonly now RNA sequencing (RNA seq) to identify the RNAs present in a biological sample
Provides information about which genes are expressed and the relative levels of expression of these genes
For example if cells are treated with a growth factor, the effect on global gene expression can be monitored and pathways that are activated by the growth factor can be determined

Question 12

Describe cDNA microarrays?

Answer 12

Typically involves converting mRNA from two samples to cDNA: one sample may be from untreated cells and one sample from treated cells
The resultant cDNA from each sample is labelled with a different fluorescent dye - with different fluorescent emissions
The labelled cDNAs are spotted on a single glass slide that has been spotted with (several thousands of) cDNA probes specific for different sequences.
Due to competitive binding between the two samples, the ratio of the fluorescence intensities for each spot is indicative of the relative abundance of the corresponding DNA probe in the two samples
Therefore data from cDNA microarrays can provide information on the relative levels of the mRNAs in each sample

Question 13

Give an example of using cDNA microarrays?

Answer 13

Used cDNA microarrays to define a signature of (mRNA) genes overexpressed for multiple types of cancer

Question 14

Describe RNA-Seq?

Answer 14

RNA sequencing (RNA-Seq) analyses complementary DNA (cDNA) copied from RNA by next-generation DNA sequencing methods
The sequence reads are then mapped onto the reference genome
This not only provides information about the genes that are expressed, but as it includes sequence information it can inform about RNA splicing
The number of sequence reads correlates with the amount of RNA (quantitative)

Question 15

Give and example of using RNA-Seq?

Answer 15

Single cell RNA-Seq of oligodendroglioma (brain tumour)

Isolated 4347 single cells from an oligodendroglioma, produced cDNA and used RNA-Seq to examine the transcriptome of each cell
Most tumour cells had a transcriptome of differentiated glial cells: astrocyte-like or oligodendrocyte-like
The transcriptome of a rare population was neuronal stem cell like that have an enhanced ability to proliferate and fuel the growth of the tumour

Question 16

Describe proteomics in cell biology?

Answer 16

Proteomics is the study of large numbers of proteins in whole organisms, tissues, cells, subcellular structures
It looks at how the levels of these change in response to external stimuli/factors
The protein content (proteome) will differ between different tissues and cell types and will reflect their specialised functions and disease state
The proteome will change in response to stimuli e.g. differentiation, virus infection, malignant transformation etc.
Proteomics can identify up to 1,000s of proteins in a biological sample and can reveal novel biological information about a sample
Mass spectrometry of trypsin digested proteins is central to most proteomics based studies

Question 17

Describe mass spectrometry in identifying proteomics?

Answer 17

Proteins are typically digested with the protease trypsin to produce peptides, which are introduced into mass spec and ionised
Trypsin cleaves after lysine
In some instances the masses of the peptides alone can be used identify to identify a protein
The peptide mass fingerprint, which is usually unique to each protein, is used to search into a database of known protein sequences and hence identify the protein

This is complicated if there are multiple proteins in a sample, but proteins can be further resolved with 2 dimensional gels
More information is often required when complex samples are analysed

Question 18

Describe mass spectrometry being used to identify more complex proteins?

Answer 18

Ionised peptides are detected by a mass spec analyser and selected one at a time to enter a collision cell
Here the selected peptide is smashed by bombardment with Argon into fragments
After collision they enter a 2nd mass spec analyser which provides mass information of the fragments
As each amino acid has different masses - the sequence of the peptide can be inferred from these fragment masses
By collating the sequences from different peptides together and by searching protein sequence databases you can determine which proteins are in your sample

Question 19

Give an example of looking at proteomics?

Answer 19

Proteomics of secretory lysosomes
Workflow:
1. Isolate secretory lysosomes from natural killer cells
Took the NK cells, homogenised, percoll gradient fractionation
2. Resolve proteins on an SDS-PAGE gel
3. Digest proteins with trypsin
4. Analyse peptides by mass spectrometry
5. Database analysis to identify proteins

221 proteins were identified and found many more proteins than we thought were present - very unbiased as it shows everything including what we are not looking for

Question 20

Describe comparative proteomics?

Answer 20

We often want to compare the protein content (proteome) of a sample before and after a specific treatment e.g. virus infection, differentiation etc
Different methods can be used in proteomics to compare samples including:
stable isotope labelling with amino acids in cell culture (SILAC)

Question 21

Describe SILAC?

Answer 21

SILAC (stable isotope labelling by/with amino acids in cell culture) is a cell culture based system that involves differential labelling of cells with amino acids, typically lysine and arginine, that have different masses i.e. 12C vs 13C labelled
It is ideal for comparing the proteomes of samples e.g. uninfected versus infected cells

There are light amino acids in one side and heavy amino acid in the other side
The samples are then mixed
Mass spectrometry will show you the relative abundance of the peptide with their different isotopes

Question 22

Give an example of using SILAC?

Answer 22

SILAC proteomic analysis of the nuclear proteome during apoptosis

Performed SILAC proteomics of JURKAT T cell line apoptosis induced with an anti-Fas antibody in

Found:
Changes in the levels of nuclear proteins, but also increased numbers of mitochondrial proteins in the nuclear fractions in apoptotic cells
Shown in follow on experiments that there was an increased association of mitochondria with nuclei in apoptotic cells