Molecular Technique Flashcards Preview

CBNS169 Human Embryology > Molecular Technique > Flashcards

Flashcards in Molecular Technique Deck (18):

Macromolecules in Cells

Nucleic Acids

A image thumb

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
  Western blot


In Situ Hybridization

It can be used to:
Determine where & when specific mRNA is expressed
Identify gene on a chromosome

Signal detection:
Colormetric probe (Enzymatic, Alkaline Phosphate)
Radioactive probe (Film)
Fluorescence probe (FISH)

Usually mRNA.
Where on a chromosome a gene is




In Situ HybridizationWhere and When Specific RNA is Expressed

(alkaline phosphatase)

Mouse E10.5

RNA Probes:

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

A image thumb

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

A image thumb

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

A image thumb

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. 

A image thumb

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.

A image thumb

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

A image thumb

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)

A image thumb

Visualize Specific Proteins using Western Blot

Proteins run on SDS PAGE are transferred to nitrocellulose membrane via current


A image thumb

Western Blot

Add primary antibody they will bind to the protein of interest 

A image thumb

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


A image thumb

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)
Western blot
SDS-PAGE: separates proteins according to size
Western Blotting: visualizes protein using antibodies