| Feature |
Nuclease
Protection Assays |
Northern
Blotting |
RT-PCR |
In Situ
Hybridization |
| Relative
and Absolute Quantitation |
Yes
|
Yes
|
Yes
|
Yes
|
| Detection
Limit (Copies of mRNA) |
4,000-5,000
|
10,000
|
1 (theoretical)
|
Medium
|
| Level
of Optimization Required |
Medium
|
Medium
|
High
|
High
|
| Sample
Size |
100 µg max
|
30 µg max
|
Not critical
|
Tissue sections
|
| Detect
Multiple Transcripts |
Yes, in same tube
|
Yes, generally requires stripping and reprobing
|
Yes, in same tube
|
No
|
| Localizes
mRNA Expression within Tissues/Cells |
No
|
No
|
No
|
Yes
|
| Mapping
Studies |
Yes
|
No
|
No
|
No
|
| Resolve
Comigrating mRNAs |
Yes
|
No
|
Yes
|
No
|
| Sizing
mRNAs |
No
|
Yes
|
No
|
No
|
| Detection
of Alternatively Spliced Transcripts |
No
|
Yes
|
No
|
No
|
| Distinguish
Between Members of Multi-Gene Families |
Yes
|
Only if they are different sizes
|
Only if they are characterized
|
No
|
| Tolerates
Partially Degraded RNA |
Yes
|
No
|
Yes
|
Yes
|
| Advantages |
- Can use up to 100 µg of sample RNA
- Ease of multiprobing
- Tolerates partially degraded RNA
|
- RNA undergoes minimal processing
- Technique can be easily evaluated at various points in
procedure
|
- Most sensitive technique available
|
- Localized mRNA within tissues
- Tolerates partially degraded RNA
|