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Northern Analysis: The Basics

lysis. Although there are a great number of protocols, techniques and commercially available kits that can be used to isolate RNA, they all share these common attributes:

  • Cellular lysis and membrane disruption
  • Inhibition of ribonuclease activity
  • Deproteinization
  • Recovery of intact RNA

Ambion provides several options for isolation of total RNA and mRNA that are compatible with a variety of cells and tissues, including bacteria, yeast, plant and animal. For a further discussion of RNA isolation options, see RNA Isolation: The Basics.


Probe Generation

Northern blots can be probed with radioactively or nonisotopically labeled RNA, DNA or oligodeoxynucleotide probes. Research at Ambion has revealed startling differences in the signal sensitivities on Northern blots achieved by three methods of probe synthesis when using standard formamide or aqueous hybridization buffers random-priming of DNA, asymmetric PCR-generated DNA and in vitro transcription of RNA. While probes for Northerns and Southerns have been historically synthesized by random-primed labeling, our results indicate that probes synthesized by asymmetric PCR are 3-5 fold more sensitive than random-primed
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