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 tissue
  • Tolerates partially degraded RNA