PMID- 37679799
OWN - NLM
STAT- MEDLINE
DCOM- 20230911
LR  - 20240503
IS  - 1756-0500 (Electronic)
IS  - 1756-0500 (Linking)
VI  - 16
IP  - 1
DP  - 2023 Sep 7
TI  - A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes.
PG  - 197
LID - 10.1186/s13104-023-06482-0 [doi]
LID - 197
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-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.
CI  - (c) 2023. BioMed Central Ltd., part of Springer Nature.
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. lrieder@emory.edu.
LA  - eng
GR  - R00 HD092625/HD/NICHD NIH HHS/United States
GR  - R35 GM142724/GM/NIGMS NIH HHS/United States
GR  - R35GM142724-01S1/NH/NIH HHS/United States
PT  - Journal Article
DEP - 20230907
PL  - England
TA  - BMC Res Notes
JT  - BMC research notes
JID - 101462768
RN  - 63231-63-0 (RNA)
RN  - 0 (Mxc protein, Drosophila)
RN  - 0 (Tumor Suppressor Proteins)
RN  - 0 (Drosophila Proteins)
SB  - IM
UOF - bioRxiv. 2023 Jun 12;:. PMID: 37398336
MH  - Animals
MH  - *Drosophila
MH  - Drosophila melanogaster/genetics
MH  - RNA/genetics
MH  - Polytene Chromosomes/genetics
MH  - In Situ Hybridization, Fluorescence
MH  - Fluorescent Antibody Technique
MH  - Tumor Suppressor Proteins
MH  - *Drosophila Proteins/genetics
PMC - PMC10486132
OTO - NOTNLM
OT  - Drosophila
OT  - Histone genes
OT  - IF
OT  - Mxc
OT  - Polytene chromosomes
OT  - RNA FISH
OT  - Transcription factor
COIS- The authors declare that they have no competing interests.
EDAT- 2023/09/08 00:42
MHDA- 2023/09/11 06:42
PMCR- 2023/09/07
CRDT- 2023/09/07 23:52
PHST- 2023/06/15 00:00 [received]
PHST- 2023/08/29 00:00 [accepted]
PHST- 2023/09/11 06:42 [medline]
PHST- 2023/09/08 00:42 [pubmed]
PHST- 2023/09/07 23:52 [entrez]
PHST- 2023/09/07 00:00 [pmc-release]
AID - 10.1186/s13104-023-06482-0 [pii]
AID - 6482 [pii]
AID - 10.1186/s13104-023-06482-0 [doi]
PST - epublish
SO  - BMC Res Notes. 2023 Sep 7;16(1):197. doi: 10.1186/s13104-023-06482-0.