2b Molecular Biology Exam Three

Analysis of RNA Expression & Localization

Constitutive expression =

gene is expressed at all times

Spatial expression =

gene is only expressed in specific tissues

Temporal expression =

gene is only expressed during a specific time in development

What are some common techniques for reading the levels of expression

Northern blot

RNase protection assays

In situ hybridization

Reverse-transcription PCRs (TR-PCR)

Edward Southern came up with a way to look for DNA sequences, it became known as a

Southern Blot

What will Northern blots do?

Measure quantities of an RNA transcript

Measure the length of an RNA transcript

What is the potential downside of Northern blots?

not sensitive to low-abundance RNAs

What is the procedure for a Northern blot?

Extract DNA

Electrophorese on agarose gel

Can look at RNA using EtBr, similar to a DNA gel

Transfer RNA to a membrane by “blotting”

Hybridize to a probe

What is included in the agarose gel for northern blots

formaldehyde

In northern blots, RNA is prone to digestion by

RNases

Why is formaldehyde added to agarose gel for Northern blots?

Helps protect against degradation by RNases

What membrane is used in northern blots?

nitrocellulose

Probe in northern blots:

Usually DNA

Can be radioactive

Could be chemiluminescent

RNA is really susceptible to

degradation

What converts RNA to DNA

Reverse Transcriptase

Where can reverse transcriptase be found?

Retroviruses

Transposons (retrotransposons)

cDNA:

You can do anything that you want with cDNA

No different than working with DNA

Usually, you want mRNA converted to cDNA

cDNA can be used for _____ or _______

PCR (RT-PCR)

qPCR (qRT-PCR)

RT-PCR detect extremely ____ abundance targets

low

______ aspect in RT-PCR can be very helpful

Quantitative

In situ hybridization is done in ______ organisms

dead

In situ can be used to locate _________

DNA or RNA

In situ can use ______

DNA prove or riboprobe

In situ has ______ technique

versatile

In situ can label probe with:

Radioactivity

Fluorophore

Antigen

How is the radioactivity technique used in situ hybridization?

(3H) detection is similar to Northerns, Southerns

How is the Fluorophore technique used in situ hybridization?

(FISH) detect with fluorescence microscopy

FISH =

Fluoroscence in situ hybridization

How is the antigen technique used in situ hybridization?

detect with enzyme-conjugated Ab to produce color change

What is the basic protocol for in situ hybridization?

Tissue is fixed

Probe is hybridized

Probe is detected

How are tissues fixed in situ hybridization?

Paraformaldehyde

Glutaraldehyde

EtOH