Exp tech in Neuro - in situ hybridisation and micro-array analysis

Question 1

In situ hybridisation

Answer 1

Localisation of mRNAs in tissue
Uses labelled DNA or RNA probe complementary to the target mRNA
Detects specific mRNA in situ, in a tissue section
Provides info on the location and amount of mRNA

Question 2

Oligonucleotides

Answer 2

Short synthetic peices of DNA 
Single stranded
Used for in situ hybridization
End labelled usjng terminal tranferase
Low cost
Most specific probe
Can synthesie sense/anti-sense control probes
Different specific activity everytime
Less sensitive than cRNA probes
Relative shortness makes cross species hybridization very unlikely.

Question 3

Complementary RNA

Answer 3

cRNA/riboprobes
Long peices of cloned sequences
Single stranded
Used for in situ hybridization
Identicle specific activity everytime
High non-specific binding
Most sensitive probe for ISH
Can generate anti-sense/sense control probes
Initial requirement for subcloning facilities
Not good for semi quantative studies

Question 4

Choice of probe used for in situ hybridization

Answer 4

Will reflect -
Sensitibity required
Reproductivity required
Facilities available
PCR fragments have been used on occasion but are artefact prone

Question 5

Likely sources of non-specific hybridization

Answer 5

Non-specific adsorption onto tissue
Hybridization to othe RNA sequences, a particular risk if there are other transcriptases highly homoogous to target mRNA.
Reduce this by optimal primer design and highly stringent conditions

Question 6

Micro array analysis

Answer 6

Assess levels of expression of thousands of mRNAs simultaneously
Designed as a screening tool
Detects which genes show altered expression after an experimental treatment or disease
Important to then confirm changes in genes usjng a more quantitative technique such as ISH