Macromolecules in Cells
Molecular Techniques Often Used in Developmental Biology
Find your gene of interest or its product
Determine if your gene of interest is transcribed and/or translated
Understand the expression pattern of your gene of interest
Techniques commonly used:
In Situ Hybridization
Reverse Transcription - Polymerase Chain Reaction (RT-PCR)
Expression Profiling: Microarray
In Situ Hybridization
It can be used to:
Determine where & when specific mRNA is expressed
Identify gene on a chromosome
Colormetric probe (Enzymatic, Alkaline Phosphate)
Radioactive probe (Film)
Fluorescence probe (FISH)
Where on a chromosome a gene is
In Situ HybridizationWhere and When Specific RNA is Expressed
Whole Mount In Situ Hybridization (WISH)
Phox2A – expressed in the dorsal ganglion regions of the nervous system
EN1 – expressed in the vertebrae and the internal gut
Uncx4- expressed in the brain and vertebrate and spinal cord
LMX1b – expressed in the brain, spinal cord and hind limb
In Situ HybridizationWhere and When Fig(alpha) is Expressed in an Ovary
Sections through mouse ovary
Probe = Radioactive Anti-sense RNA to Fig a
Where is Fig (alpha) located?
Visualized on a film
Cluster of the white is the fig alpha and is expressed in the primary oocytes
Bright white is probably the primordial follicle
Knockout does not show any expression
In Situ HybridizationHow many copies of a specific chromosome are in a cell?
FISH: fluorescence in situ hybridization
Used here to determine number of copies of chromosome 21 in a cell.
Cell has been treated with a fluorescent probe that binds to a gene on chromosome 21.
How many copies of chromosome 21 are in each cell?
FISH technique stains with DAPI as well
Blue stain on nuclei, different color used for each chromosome
In Situ Hybridization pros/cons
Gives cellular localization of specific mRNA and genes being expressed or the number of chromosomes in a cell
Can be used on whole organisms, tissue sections, cells, or chromosomes
May be limited by the abundance of the mRNA that is being probed
Reverse Transcription – Polymerase Chain ReactionAmplifies DNA or mRNA
Amplifies and detects DNA sequence of interest
Only works with DNA
What if you have mRNA of interest?
First make cDNA from mRNA
then do PCR
mRNA => cDNA => RT-PCR
This method can detect the expression of a specific mRNA (semiquantitative).
Denature DNA with heat, add primer (which binds to the two strands). Replicates until you get as much as you need.
Reverse Transcription – Polymerase Chain Reaction pros/cons
Allows for amplification of the DNA/mRNA sample
Few copies of the transcript can be used to amplify and see the signal
RT-PCR will not tell the cellular location of the genes being expressed
Expression Profiling: DNA Microarray
Can be used to quantify the expression of many genes simultaneously
Similar to RT-PCR except thousands of genes can be studied at one time
Isolate mRNA, label with fluorescent probe. Incubate cell types.
Green is in control, red is experimental yellow is both.
DNA Microarray pros/cons
Allows for analysis of multiple genes or the entire genome simultaneously
Tremendous read out of genes expressed
Can be Expensive
Is mRNA translated?
DNA RNA protein
Method usually used to study translation of mRNA into protein is SDS-PAGE followed by Western Blotting
SDS: Sodium dodecyl sulfate
PAGE: Polyacrylamide gel electrophoresis
SDS-PAGE Separates Proteins from Each Other
Sample of proteins
Proteins are treated with an ionic detergent (SDS) that denatures & unwinds them, and coats them with a negative charge
Mixture is loaded into a gel that separates proteins by size
Run a current through your gel and the proteins will separate by size
SDS-PAGE Separates Proteins from Each Other cont.
Now, you have your SDS gel with separated proteins
If you want to visualize the proteins on your gel, you can stain using a coomassie blue stain. (image right)
Visualize Specific Proteins using Western Blot
Proteins run on SDS PAGE are transferred to nitrocellulose membrane via current
Add primary antibody they will bind to the protein of interest
Western Blot cont.
Add secondary antibody bind to the primary antibody.
Secondary antibody must be directed against the species in which the primary antibody was made.
Secondary antibody has a luciferase reporter and cleaves luciferin which generates light and is detected using film (which is the black band)
Presence or absence and the amount of the protein can be determined
summary of all Molecular Techniques
DNA or mRNA expression
In Situ Hybridization
Used on embryos, tissues, cells to visualize mRNA expression
Can be used on chromosomes to locate specific genes or determine number of copies of a chromosome
Polymerase Chain Reaction (PCR)
PCR: Amplifies DNA for detection
RT-PCR: Transcribes mRNA into cDNA, then amplifies specific cDNA
Expression profiling: Microarray
Examines expression of multiple genes from a single tissue/cell type simultaneously
Protein expression (mRNA translation)
SDS-PAGE: separates proteins according to size
Western Blotting: visualizes protein using antibodies