PMID- 37398336
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20240503
DP  - 2023 Jun 12
TI  - A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes.
LID - 2023.06.12.544616 [pii]
LID - 10.1101/2023.06.12.544616 [doi]
AB  - OBJECTIVES: Investigating protein-DNA interactions is imperative to understanding 
      fundamental concepts such as cell growth, differentiation, and cell development 
      in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA 
      binding profiles of transcription factors; however this assay can be expensive, 
      time-consuming, may not be informative for repetitive regions of the genome, and 
      depend heavily upon antibody suitability. Combining DNA fluorescence in situ 
      hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive 
      approach which has historically been used to investigate protein-DNA interactions 
      in individual nuclei. However, these assays are sometimes incompatible due to the 
      required denaturation step in DNA FISH that can alter protein epitopes, hindering 
      primary antibody binding. Additionally, combining DNA FISH with IF may be 
      challenging for less experienced trainees. Our goal was to develop an alternative 
      technique to investigate protein-DNA interactions by combining RNA FISH with IF. 
      RESULTS: We developed a hybrid RNA FISH and IF protocol for use on Drosophila 
      melanogaster polytene chromosome spreads in order to visualize colocalization of 
      proteins and DNA loci. We demonstrate that this assay is sensitive enough to 
      determine if our protein of interest, Multi-sex combs (Mxc), localizes to 
      single-copy target transgenes carrying histone genes. Overall, this study 
      provides an alternative, accessible method for investigating protein-DNA 
      interactions at the single gene level in Drosophila melanogaster polytene 
      chromosomes.
FAU - Gilbonio, Hannah E
AU  - Gilbonio HE
AD  - Department of Biology, Emory University, Atlanta, GA, USA.
FAU - Puckett, Gwyn L
AU  - Puckett GL
AD  - Piedmont Virginia Community College, Charlottesville, VA, USA.
FAU - Nguyen, Erica
AU  - Nguyen E
AD  - Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
FAU - Rieder, Leila E
AU  - Rieder LE
AD  - Department of Biology, Emory University, Atlanta, GA, USA.
LA  - eng
GR  - R00 HD092625/HD/NICHD NIH HHS/United States
GR  - R35 GM142724/GM/NIGMS NIH HHS/United States
PT  - Preprint
DEP - 20230612
PL  - United States
TA  - bioRxiv
JT  - bioRxiv : the preprint server for biology
JID - 101680187
UIN - BMC Res Notes. 2023 Sep 7;16(1):197. PMID: 37679799
PMC - PMC10312655
COIS- Competing interests None.
EDAT- 2023/07/03 13:05
MHDA- 2023/07/03 13:06
PMCR- 2023/06/30
CRDT- 2023/07/03 11:45
PHST- 2023/07/03 13:06 [medline]
PHST- 2023/07/03 13:05 [pubmed]
PHST- 2023/07/03 11:45 [entrez]
PHST- 2023/06/30 00:00 [pmc-release]
AID - 2023.06.12.544616 [pii]
AID - 10.1101/2023.06.12.544616 [doi]
PST - epublish
SO  - bioRxiv [Preprint]. 2023 Jun 12:2023.06.12.544616. doi: 
      10.1101/2023.06.12.544616.