RNA expression Flashcards
Central dogma of cell biology
Gene expression is production of a functional RNA or protein from the genetic information encoded by the genes
Differential expression of genes results in different cells, tissues and organs
Central dogma - DNA makes RNA makes protein
Can measure activity of a gene at different levels:
- Transcription (RNA)
- Translation
- Molecular interactions - proteins don’t work alone
Gene expression
Want to know:
- How much there is
- Where it is
- What it’s interacting with
Method that can be used:
- RNA/DNA
- PCR-based
- Hybridisation-based
- Protein
- Immunological (antibody)-based
- Fusion proteins
RNA expression and localisation
PCR-based methods
- RT-PCR
Hybridisation-based techniques
- RNA expression
- Northern Blot
- Microarray
- RNA localisation
- FLuorescene In situ hybridisation
RNA expression - Northern Blot
Steps:
1. RNA is separated by electrophoresis (based on size)
2. RNA is transferred to a membrane to allow for blotting
3. A gene-specific probe is added and hybridise to the target sequence
RNA expression - microarrays
Oligonucleotides are attached to a spot on a chip
Each spot has a different oligonucleotide, corresponding to a specific gene
RNA is prepared from a source and fluorescently-labelled cDNA is made from the RNA
Fluorescent cDNA is applied to the chip and allowed to hybridise
Microarrays measure relative mRNA levels
Not easy to quantity absolute values of mRNA levels
Better for assaying relative levels
Different fluorescent tags for cDNA from different sources
RNA localisation - Fluorescent In situ hybridisation
Fluorescent In situ hybridisation = FISH for short
Principle of hybridisation is similar to northern blot
Probe is often labelled with fluorescent marker and visualised using microscope
DNA/RNA localisation within chromosome, a cell, an organ, the whole organism
Correct RNA localisation can be vital for correct development
Protein-specific antibodies
Conjugate - specific to the application
Secondary antibody - recognises the primary antibody
Protein-specific antibody - primary antibody
Western blotting
Steps:
1. Proteins are separated by electrophoresis (based on size)
2. Then transferred to a membrane
3. Detection is using a protein-specific antibody and a labelled secondary antibody
Immunofluorescence
Not live imaging - a snapshot in time (cells usually need to be fixed and prepared in quite harsh way)
Secondary antibody usually conjugated to a fluorescent molecule - imaged using microscope
Use different fluorophores - can see more than one molecule at a time
Living imaging of protein localisation
Fuse to a protein that is easy to visualise
Fuse the two protein-coding sequences (CDS) - visualise the reporter to assess localisation
Fluorescent proteins are commonly used - visualised by microscopy
Other fusion proteins exist
Fluorescent fusion proteins
Green Fluorescent Protein was one of the earliest
Now there are lots from different sources, so can look at more than one protein at a time
Can combine different techniques
Use GFP as a reporter gene for one protein
And an antibody to visualise another protein
Shows colocalization of the two proteins - may be functionally significant
Report genes
Reporter genes:
- Easy to visualise e.g. like GFP
- Or easy to assay e.g. luciferase and beta-galactosidase
Common uses of luciferase and beta-galactosidase
- Clone next to the promoter for Your Favourite Gene (YFG)
- Put recombinant promoter-reporter into cells
- Investigate different growth conditions, role of transcriptional activators/ repressors etc
- The reporter protein is an easily measurable proxy for expression of YFG
Expression of reporter gene = expression of YFG
Analysis of molecular interaction
Tools for analysing interactions - proteins are rarely functional alone, usually function as part
