Comparing mRNA levels using in situ hybridization of a target gene and co-stain

Methods. 2014 Jun 15;68(1):233-41. doi: 10.1016/j.ymeth.2014.01.003. Epub 2014 Jan 13.


In situ hybridization is an important technique for measuring the spatial expression patterns of mRNA in cells, tissues, and whole animals. However, mRNA levels cannot be compared across experiments using typical protocols. Here we present a semi-quantitative method to compare mRNA levels of a gene across multiple samples. This method yields an estimate of the error in the measurement to allow statistical comparison. Our method uses a typical in situ hybridization protocol to stain for a target gene and an internal standard, which we refer to as a co-stain. As a proof of concept, we apply this method to multiple lines of transgenic Drosophila embryos, harboring constructs that express reporter genes to different levels. We generated this test set by mutating enhancer sequences to contain different numbers of binding sites for Zelda, a transcriptional activator. We demonstrate that using a co-stain with in situ hybridization is an effective method to compare mRNA levels across samples. This method requires only minor modifications to existing in situ hybridization protocols and uses straightforward analysis techniques. This strategy can be broadly applied to detect quantitative, spatially resolved changes in mRNA levels.

Keywords: Drosophila melanogaster; In situ hybridization; Transcriptional activator; Vielfaltig; Zelda; mRNA levels.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Base Sequence
  • Developmental Biology / methods
  • Drosophila
  • Drosophila Proteins / biosynthesis*
  • Gene Expression Regulation, Developmental
  • In Situ Hybridization / methods*
  • Nuclear Proteins
  • RNA, Messenger / biosynthesis*
  • Transcription Factors / biosynthesis*


  • Drosophila Proteins
  • Nuclear Proteins
  • RNA, Messenger
  • Transcription Factors
  • zld protein, Drosophila