Simultaneous detection of the subcellular localization of RNAs and proteins in cultured cells by combined multicolor RNA-FISH and IF.
Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) are sensitive techniques used for detecting nucleic acids and proteins in cultured cells. However, these techniques are rarely applied together, and standard protocols are not readily compatible for sequential application on the same specimen. Here, we provide a user-friendly step-by-step protocol to perform multicolor RNA-FISH in combination with IF to simultaneously detect the subcellular localization of distinct RNAs and proteins in cultured cells. We demonstrate the use of our protocol by analyzing changes in the subcellular distribution of RNAs and proteins in cells exposed to a variety of stress conditions.
Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, United States.
Cindy
C
Meyer
Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, United States.
Aitor
A
Garzia
Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, United States. Electronic address: ttuschl@rockefeller.edu.
Thomas
T
Tuschl
D000906:Antibodies / Q000737:chemistry; D018053:Arsenites / Q000633:toxicity; D001312:Aurintricarboxylic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D020365:RNA Helicases / Q000235:genetics* / Q000378:metabolism; D000067776:RNA Recognition Motif Proteins / Q000235:genetics* / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics* / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000235:genetics* / Q000378:metabolism; D017670:Sodium Compounds / Q000633:toxicity; D013312:Stress, Physiological; D019284:Thapsigargin / Q000494:pharmacology
D000906:Antibodies; D018053:Arsenites; D015345:Oligonucleotide Probes; D000075223:Poly-ADP-Ribose Binding Proteins; D000067776:RNA Recognition Motif Proteins; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D017670:Sodium Compounds; D049452:Green Fluorescent Proteins; D001312:Aurintricarboxylic Acid; C017947:sodium arsenite; D019284:Thapsigargin; C017037:aurin; D006861:Hydrogen Peroxide; D004265:DNA Helicases; C116070:G3BP1 protein, human; D020365:RNA Helicases
Antibody; FISH; GFP; Immunofluorescence; Nucleic acids; Stress granule
Nat Rev Genet. 2014 Dec;15(12 ):829-45
25365966
J Exp Med. 1950 Jan 1;91(1):1-13
15395569
Proc Natl Acad Sci U S A. 1969 Jun;63(2):378-83
4895535
Peptides. 2007 Feb;28(2):226-34
17207557
Exp Cell Res. 2008 Feb 1;314(3):543-53
18164289
Trends Immunol. 2014 Sep;35(9):420-8
25153707
Brain Res. 2014 Oct 10;1584:52-8
25108040
J Cell Biol. 1999 Dec 27;147(7):1431-42
10613902
Exp Cell Res. 1973 Apr;78(2):360-6
4633335
Mol Cell. 2009 Dec 25;36(6):932-41
20064460
Mol Biol Cell. 2012 Sep;23(18):3499-510
22833567
Science. 1994 Feb 11;263(5148):802-5
8303295
Methods Enzymol. 2013;529:99-124
24011039
Trends Biochem Sci. 2008 Mar;33(3):141-50
18291657
J Cell Biol. 2005 Jun 20;169(6):871-84
15967811
Appl Microbiol Biotechnol. 2003 Jan;60(5):523-33
12536251
Methods Mol Biol. 2014;1211:171-87
25218385
J Cell Sci. 2009 Feb 15;122(Pt 4):563-73
19193871
Nucleic Acids Res. 1999 Nov 15;27(22):4436-43
10536153
J Clin Invest. 2013 Jun;123(6):2694-702
23728175
Nat Biotechnol. 2004 Dec;22(12):1567-72
15558047
Mol Cell Biol. 2014 Jun;34(11):2003-16
24662051
Virology. 2013 Feb 5;436(1):1-7
23102969
Gene. 1992 Feb 15;111(2):229-33
1347277
J Cell Biol. 2015 Apr 13;209(1):73-84
25847539
Cell. 1986 Oct 10;47(1):141-52
3093084
Nat Biotechnol. 1999 Oct;17(10):969-73
10504696
Biochem Biophys Res Commun. 2012 Jul 13;423(4):763-9
22705549
Dev Biol. 1998 Jul 15;199(2):245-9
9698444
Biochim Biophys Acta. 2015 Jul;1849(7):861-70
25482014
J Mol Diagn. 2011 May;13(3):282-8
21497290
Nat Rev Mol Cell Biol. 2005 Nov;6(11):885-91
16167053
Chromosoma. 1989 Aug;98(2):81-5
2476281
Biochemistry. 2004 Oct 26;43(42):13233-41
15491130
J Cell Comp Physiol. 1962 Jun;59:223-39
13911999
Trends Biotechnol. 2012 Jan;30(1):8-16
21924508
Nat Methods. 2006 Jan;3(1):27-9
16369549