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Comparison of Different Methods of mRNA Quantitation


FeatureNuclease Protection AssaysNorthern BlottingRT-PCRIn Situ HybridizationRelative 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 g max
30 g max
Not critical
Tissue sections
Detect Multiple Transcripts
Yes, in same tube
Yes, ge nerally 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 g 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

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