Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

Amrita D. Chaudhuri, Sowmya V Yelamanchili, Howard S Fox

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure.

Original languageEnglish (US)
Article number160
JournalFrontiers in Cellular Neuroscience
Issue numberSEP
DOIs
StatePublished - Sep 23 2013

Fingerprint

Fluorescence In Situ Hybridization
MicroRNAs
Paraffin
Formaldehyde
Nucleic Acid Probes
RNA
Small Untranslated RNA
In Situ Hybridization
Digestion
Buffers
Proteins
Peptide Hydrolases
Gene Expression
Neurons
Messenger RNA
Temperature

Keywords

  • Brain
  • FFPE
  • LNA
  • Neuron
  • TSA

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience

Cite this

@article{5585bbca9aee4a2b8c6b92391e9ad7c3,
title = "Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers",
abstract = "Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure.",
keywords = "Brain, FFPE, LNA, Neuron, TSA",
author = "Chaudhuri, {Amrita D.} and Yelamanchili, {Sowmya V} and Fox, {Howard S}",
year = "2013",
month = "9",
day = "23",
doi = "10.3389/fncel.2013.00160",
language = "English (US)",
journal = "Frontiers in Cellular Neuroscience",
issn = "1662-5102",
publisher = "Frontiers Research Foundation",
number = "SEP",

}

TY - JOUR

T1 - Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

AU - Chaudhuri, Amrita D.

AU - Yelamanchili, Sowmya V

AU - Fox, Howard S

PY - 2013/9/23

Y1 - 2013/9/23

N2 - Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure.

AB - Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure.

KW - Brain

KW - FFPE

KW - LNA

KW - Neuron

KW - TSA

UR - http://www.scopus.com/inward/record.url?scp=84885029172&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84885029172&partnerID=8YFLogxK

U2 - 10.3389/fncel.2013.00160

DO - 10.3389/fncel.2013.00160

M3 - Article

JO - Frontiers in Cellular Neuroscience

JF - Frontiers in Cellular Neuroscience

SN - 1662-5102

IS - SEP

M1 - 160

ER -