RNA & Transcriptomics 2

describe the steps for RNA sequencing

1. isolate RNA from sample

2. fragment RNA into short segments

3. convert RNA fragments into cDNA

4. ligate sequencing adapters & amplify

5. perform NGS sequencing

6. map sequencing reads to the transcriptome/genome

what is a gene expression profile

the “on” & “off” states of all of a cells genes

• each cell type has a unique profile

• genes expressed by a cell can give insight into the function & fate of the cell

what are some different types of gene expression profiles

• profile distinct cell subsets (e.g. immune cells)

• profile temporal changes

• profile response to stimuli (infection, activation)

• profile healthy vs diseased (tumours, inflamed)

how do we profile gene expression

1. isolate mRNA from cells

   • rare cells make it hard to get enough RNA

2. consider the question we want to answer

   • cell type, disease state, timing

what are some things to look for when profiling

• is a gene expressed at all within the cell type?

• is the gene expression level changed at all?

  • higher or lower

what does gene expression tell us

isolating mRNA from cells at a given time, allows us to examine which genes are transcribed at a specific time

• only a snapshot of gene expression at a specific time

what controls the gene expression profile in cells

cell linage-specific expression of transcription factors

• gene expression is controlled by binding of transcription factors

what is important to remember about gene expression profiling

think of it as taking the average, this means that difference between individual cells can be masked making them harder to identify

when would you use bulk or scRNA-seq

bulk RNA-seq:

• cheap

• used in most cases

• broad analysis

scRNA-seq:

• expensive

• precise

what are some pros & cons of scRNA-Seq

PROS:

• finely detailed map of cell population

• identify rare cell types & relationships

CONS:

• expensive

• misses lowly expressed genes

• trickly to analyze

what is some general information about cancer

• progressive (cell → tumour → metastasis)

• multistep process, requires multiple mutations to reach the tumour state

what 2 cell types, when mutated, can give rise to cancer

• proto-oncogenes

• tumor suppressor genes

what are proto-oncogenes

a group of genes that cause normal cell to become cancerous when they are mutated

• mutation are typically dominant in nature

• mutated proto-oncogene = oncogene

• often code for cell division, death, differentiation

what type of proteins do tumor suppressor genes code for

• cell adhesion/recognition

  • mutations cause cells to lose adhesion & spread

• enzymes for DNA repair

  • mutations are more prominent

• inhibit cell division

  • mutations causes rapid division

how can gene expression profiling identify cancer

• identify whether tumor suppressor/oncogenes are lost/gained is critical

• identify novel drivers of specific cancers

how can difference between tumors and normal tissue be found

comparative transcriptomics

• can identify decreases/increases in certain driver genes

• identifies pathways & downstream changes

what is tumor heterogeneity

due to the speed of tumor growth, different parts of the tumor can growth different mutations

• hence 1 medication may only kill one part of the tumor but not the rest