title: The importance of tissue specificity for RNA-seq: highlighting the errors of composite structure extractions.
abstract: BACKGROUND
  A composite biological structure, such as an insect head or abdomen, contains many internal structures with distinct functions. Composite structures are often used in RNA-seq studies, though it is unclear how expression of the same gene in different tissues and structures within the same structure affects the measurement (or even utility) of the resulting patterns of gene expression. Here we determine how complex composite tissue structure affects measures of gene expression using RNA-seq.
  
  
  RESULTS
  We focus on two structures in the honey bee (the sting gland and digestive tract) both contained within one larger structure, the whole abdomen. For each of the three structures, we used RNA-seq to identify differentially expressed genes between two developmental stages, nurse bees and foragers. Based on RNA-seq for each structure-specific extraction, we found that RNA-seq with composite structures leads to many false negatives (genes strongly differentially expressed in particular structures which are not found to be differentially expressed within the composite structure). We also found a significant number of genes with one pattern of differential expression in the tissue-specific extraction, and the opposite in the composite extraction, suggesting multiple signals from such genes within the composite structure. We found these patterns for different classes of genes including transcription factors.
  
  
  CONCLUSIONS
  Many RNA-seq studies currently use composite extractions, and even whole insect extractions, when tissue and structure specific extractions are possible. This is due to the logistical difficultly of micro-dissection and unawareness of the potential errors associated with composite extractions. The present study suggests that RNA-seq studies of composite structures are prone to false negatives and difficult to interpret positive signals for genes with variable patterns of local expression. In general, our results suggest that RNA-seq on large composite structures should be avoided unless it is possible to demonstrate that the effects shown here do not exist for the genes of interest.
authors.affiliation: Department of Entomology, University of California, Davis, 1 Shields Ave, Davis, CA 95616, USA. brnjohnson@ucdavis.edu.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Johnson
authors.forename: Joel
authors.initials: J
authors.lastname: Atallah
authors.forename: David C
authors.initials: DC
authors.lastname: Plachetzki
mesh_terms: D000818:Animals; D001514:Bee Venoms / Q000235:genetics / Q000378:metabolism; D001516:Bees / Q000235:genetics / Q000378:metabolism; D005188:False Negative Reactions; D041981:Gastrointestinal Tract / Q000378:metabolism; D020869:Gene Expression Profiling; D017344:Genes, Insect; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics; D017423:Sequence Analysis, RNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: D001514:Bee Venoms; D019476:Insect Proteins; D014157:Transcription Factors