Untitled Flashcards Set

Analysis of Gene Expression

1.RNA expression and localisation

2.Protein expression, localisation

3.Reporter genes

4.Analysis of interactions

RNA

  • Expression levels

  • Different isoforms

  • Localisation (cell —> organism)

Protein

  • Expression levels

  • Different isoforms

  • Post-translational modifications

  • Localisation

Interactions with other molecules

What we’d like to know?

How much is there? Where is it? What is it interacting with?

Methods we can use:

  • DNA / RNA

    • PCR-based

    • Hybridisation-based

    • Reporter genes

  • Protein

    • Immunological (antibody)-based

    • Fusion proteins

RNA Expression & Localisation

PCR-BASED METHODS

  • RT-PCR (quantitative or non-quantitative)


non-quantitative:


mRNA (isoform 1)


mRNA (isoform 2)

  • This can give information about relative amounts

  • Or can potentially distinguish isoforms by size/RE digest

HYBRIDISATION-BASED TECHNIQUES

Based on properties of DNA/RNA, complementary sequences will hybridise to each other

If you have a sequence that you are interested in (RNA/cDNA/DNA), you can use that sequence to make a “probe”

Probe: DNA sequence that is complementary to the RNA sequence you want to analyse (labelled in some way that we can analyse) - written in 5’ to 3’

This binds in a sequence-specific manner using base-pairing rules


  • RNA expression:

    • Northern Blot

    • Microarray

  • RNA localisation:

    • Fluorescence In situ hybridisation

Northern Blot


  1. RNA is separated by electrophoresis (based on size) = will see major bands of rRNA


  1. RNA is transferred to a membrane to allow for blotting


  1. A gene-specific probe is added and hybridises to the target sequence

  • Can tell you about abundance, size, isoforms, expression in different tissues/cell types

  • Limited to a single gene each time

Microarrays

  • Oligonucleotides (short DNA molecules) are attached to a spot (lots of the same oligonucleotide) 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 = RNA is not very stable

  • Fluorescent cDNA is applied to the chip and allowed to hybridise

Lots of mRNA —> lots of cDNA —> lots of fluorescence

Microarrays measure relative mRNA levels

  • Difficult to quantify absolute values of mRNA levels

  • Better for assaying relative levels

  • Different fluorescent tags for cDNA from different sources


How do we compare levels of transcription?





Black: No cDNA from either source Yellow: Equal cDNA from both sources Green: More cDNA from tumour cells Red: more cDNA from normal cells

Fluorescence In situ hybridisation

(FISH)

  • Principle of hybridisation is same as for Northern blot

  • Probe is labelled with a fluorescent marker and visualised using microscopy

    • Localisation of RNA

    • Or many RNAs (multiple probes)

    • Or RNA and proteins

  • Can also be used to localise DNA = Localisation of RNA to the right place can be vital in development

REPORTER GENES

Easy to visualise (fluorescent) or assay (enzyme)

Enzymatic reporter genes: luciferase and beta-galactosidase

  1. Clone next to a promoter of interest

  2. Put recombinant promoter-reporter into cells

  3. Investigate different growth conditions, role of transcriptional activators / repressors, etc.

  4. The reporter protein is an easily measurable proxy for promoter activity

↑ Expression of reporter gene = ↑ promoter activity

Eg. Luciferase assay


  • Need an internal control (another fluorescent molecule)

  • Better for relative expression levels (unless using endogenous promoter)

  • Doesn’t have to be a transcriptional protein – could be some sort of growth condition or drug

Protein Expression & Localisation

PROTEIN-SPECIFIC ANTIBODIES

  • Conjugate (specific to the application and allows detection)

  • Secondary antibody (recognises the primary antibody, usually raised to the species)

  • Protein-specific antibody (primary antibody)

Western Blotting


  1. Proteins are separated by electrophoresis (based on size)


  1. Protein is then transferred to a membrane


  1. Detection is using a protein-specific antibody and a labelled secondary antibody

Immunofluorescence

  • Secondary antibody is usually conjugated to a fluorescent molecule – imaged using microscopy

  • Use different fluorophores – can see more than one molecule at a time

  • Not live imaging – a snapshot in time (cells usually need to be fixed and prepared in quite harsh ways)


Fluorescent fusion proteins

  • Green Fluorescent Protein was one of the earliest

  • Now there are lots of them (from different sources), so we can look at more than one protein at a time


LIVE IMAGING OF PROTEIN LOCALISATION

  • Use molecular cloning:

    • Fuse the Coding Sequence (CDS) for your protein of interest (Protein X) to that of a fluorescent protein (GFP):


  • Fluorescent proteins can be directly visualised

  • (Other fusion proteins exist for different applications)

COMBINING TECHNIQUES


GFP as a reporter gene for one protein

An antibody to visualise another protein

Shows co-localisation of the two proteins - this may be functionally significant

Analysis of Molecular Interactions

Tools for analysing interactions:

  • Proteins rarely function alone. They usually function as part of large complexes with other molecules

  • Identifying a protein’s interacting partners might be very useful functional information

  • Protein-protein interactions:

    • Pull-down assay

    • Immunoprecipitation

    • Yeast two-hybrid

  • Protein-DNA interactions:

    • Chromatin Immunoprecipitation

PROTEIN-PROTEIN INTERACTIONS

Pull-down assay

  • A way of analysing protein interactions in vitro

  • Uses fusion proteins

    • Affinity for a specific ligand = easy to purify

    • Glutathione S-Transferase (GST) is commonly used

    • Binds reduced glutathione (GSH)

  1. Make a recombinant DNA molecule:


  1. Insert recombinant DNA into bacteria to make protein, which makes a cell lysate (GST and protein X)

  2. Bind GST (and protein X) to the affinity ligand

  3. Wash away any unwanted stuff —> purified recombinant protein

  4. Investigate what can bind to Protein X.

Immunoprecipitation

  1. Almost identical technique and method to a pulldown.

  2. Uses antibodies to directly bind Protein X.

  3. See what interacts with it (co-immunoprecipitation).

Advantages:

  • Native protein can give better results (less background, fewer false positive)

Disadvantages:

  • Antibodies are not easy to make: relatively expensive, not readily available, very variable, and limited supply.

  • Need to recognise native protein

To analyse interacting proteins, use western blotting or mass spec


Yeast two-hybrid

Uses fusion proteins

  • Transcription factors are modular so have separate domains for DNA-binding and transcription activation

    • Separate —> no transcription activation

    • Bring together —> transcription activation


  • DNA Binding Domain fused to a “bait”

  • Transcription Activation domain fused to a “prey”

  • If bait and prey interact, the reporter gene is transcribed

PROTEIN-DNA

Chromatin Immunoprecipitation (ChIP)

  • Used to study interaction of proteins with DNA

  • Use an antibody to purify your protein of interest (could also use a fusion protein too)

  • Assay which DNA molecules are associated with your protein


  1. Cross-link DNA-protein first (or the interaction will not survive harsh purification conditions)

  2. Need to break up DNA (interested in local interactions)

  3. Immunoprecipitation – anything not bound to antibody remains in solution

  4. DNA purification

  5. DNA analysis – qPCR; microarray; ChIP-seq (uses Next Generation Sequencing to give a genome-wide view of the DNA sequences)

SUMMARY

How much detail?

  • The basic principle of the method

    • PCR? Antibodies?

  • What molecule does it detect?

    • RNA? Protein?

  • What does it tell us about that molecule?

    • Where it is? How much there is?

































































What do i need to know?

  • Explain how hybridisation-based methods work when analysing RNA and design a suitable probe from a sequence.

  • Name methods that use hybridisation to analyse RNA (Norther blot, microarray, FISH). Explain the basic method and what each method can measure.

  • Explain how a reporter gene works.

  • Explain how antibodies work when analysing proteins, including the role of secondary antibodies.

  • Name methods that use antibodies (western blot, immunofluorescence). Explain the basic method and what each method can measure.

  • Explain how a fusion protein works.

  • Name and describe methods for analysing protein-protein interactions (pulldown, yeast two hybrid, immunoprecipitation) and protein-DNA interactions (ChIP).