Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR

Ron S. Broide, Alain Trembleau, Julie A. Ellison, Judith Cooper, David Lo, Warren G. Young, John Morrison, Floyd E. Bloom

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

In situ hybridization (ISH) is an essential technique for mapping gene expression in the brain. Although many ISH protocols provide for quantitative analysis of individual mRNAs in different brain regions or across experimental conditions, this technique has lacked the necessary standardization for quantitative comparisons between different mRNA transcripts. We have developed a standardized quantitative ISH (SQuISH™) protocol that utilizes multiple radioactive oligonucleotide probes, providing for increased sensitivity, decreased background and accurate comparison of relative mRNA levels. We evaluated the SQuISH™ protocol against a riboprobe-based ISH procedure by comparing the mRNA expression levels in the brain for two transcripts, insulin receptor substrate p53 (IRSp53) and Calsenilin. The results of these two methods were then validated by real-time quantitative PCR. Both protocols exhibited identical mRNA expression patterns for IRSp53 and Calsenilin. In three brain regions analyzed, the levels of IRSp53 mRNA expression were ∼1.5-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure, although the relative abundance in regional expression levels was similar between the two methods. In contrast, the levels of Calsenilin mRNA expression were 10-17-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure and the relative abundance in regional expression levels was different. When compared to the real-time PCR results, the SQuISH™ method showed almost identical relative levels of IRSp53 to Calsenilin mRNA in all three brain regions analyzed, while the riboprobe-based procedure showed a completely opposite trend. These results support the accuracy of the SQuISH™ protocol for determining relative mRNA levels in the brain.

Original languageEnglish (US)
Pages (from-to)211-222
Number of pages12
JournalBrain Research
Volume1000
Issue number1-2
DOIs
StatePublished - Mar 12 2004
Externally publishedYes

Fingerprint

Oligonucleotide Probes
In Situ Hybridization
Real-Time Polymerase Chain Reaction
Kv Channel-Interacting Proteins
Messenger RNA
Insulin Receptor
Brain
Gene Expression

Keywords

  • Calsenilin
  • Insulin receptor substrate p53
  • Neurotransmitters, modulators, transporters, and receptors
  • Oligonucleotide probe
  • Quantitative in situ hybridization
  • Real-time quantitative PCR
  • Signal transduction: gene expression

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology

Cite this

Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR. / Broide, Ron S.; Trembleau, Alain; Ellison, Julie A.; Cooper, Judith; Lo, David; Young, Warren G.; Morrison, John; Bloom, Floyd E.

In: Brain Research, Vol. 1000, No. 1-2, 12.03.2004, p. 211-222.

Research output: Contribution to journalArticle

Broide, Ron S. ; Trembleau, Alain ; Ellison, Julie A. ; Cooper, Judith ; Lo, David ; Young, Warren G. ; Morrison, John ; Bloom, Floyd E. / Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR. In: Brain Research. 2004 ; Vol. 1000, No. 1-2. pp. 211-222.
@article{2da86b7b91374a96a026d8e446f9e3f9,
title = "Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR",
abstract = "In situ hybridization (ISH) is an essential technique for mapping gene expression in the brain. Although many ISH protocols provide for quantitative analysis of individual mRNAs in different brain regions or across experimental conditions, this technique has lacked the necessary standardization for quantitative comparisons between different mRNA transcripts. We have developed a standardized quantitative ISH (SQuISH™) protocol that utilizes multiple radioactive oligonucleotide probes, providing for increased sensitivity, decreased background and accurate comparison of relative mRNA levels. We evaluated the SQuISH™ protocol against a riboprobe-based ISH procedure by comparing the mRNA expression levels in the brain for two transcripts, insulin receptor substrate p53 (IRSp53) and Calsenilin. The results of these two methods were then validated by real-time quantitative PCR. Both protocols exhibited identical mRNA expression patterns for IRSp53 and Calsenilin. In three brain regions analyzed, the levels of IRSp53 mRNA expression were ∼1.5-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure, although the relative abundance in regional expression levels was similar between the two methods. In contrast, the levels of Calsenilin mRNA expression were 10-17-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure and the relative abundance in regional expression levels was different. When compared to the real-time PCR results, the SQuISH™ method showed almost identical relative levels of IRSp53 to Calsenilin mRNA in all three brain regions analyzed, while the riboprobe-based procedure showed a completely opposite trend. These results support the accuracy of the SQuISH™ protocol for determining relative mRNA levels in the brain.",
keywords = "Calsenilin, Insulin receptor substrate p53, Neurotransmitters, modulators, transporters, and receptors, Oligonucleotide probe, Quantitative in situ hybridization, Real-time quantitative PCR, Signal transduction: gene expression",
author = "Broide, {Ron S.} and Alain Trembleau and Ellison, {Julie A.} and Judith Cooper and David Lo and Young, {Warren G.} and John Morrison and Bloom, {Floyd E.}",
year = "2004",
month = "3",
day = "12",
doi = "10.1016/j.brainres.2003.11.069",
language = "English (US)",
volume = "1000",
pages = "211--222",
journal = "Brain Research",
issn = "0006-8993",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR

AU - Broide, Ron S.

AU - Trembleau, Alain

AU - Ellison, Julie A.

AU - Cooper, Judith

AU - Lo, David

AU - Young, Warren G.

AU - Morrison, John

AU - Bloom, Floyd E.

PY - 2004/3/12

Y1 - 2004/3/12

N2 - In situ hybridization (ISH) is an essential technique for mapping gene expression in the brain. Although many ISH protocols provide for quantitative analysis of individual mRNAs in different brain regions or across experimental conditions, this technique has lacked the necessary standardization for quantitative comparisons between different mRNA transcripts. We have developed a standardized quantitative ISH (SQuISH™) protocol that utilizes multiple radioactive oligonucleotide probes, providing for increased sensitivity, decreased background and accurate comparison of relative mRNA levels. We evaluated the SQuISH™ protocol against a riboprobe-based ISH procedure by comparing the mRNA expression levels in the brain for two transcripts, insulin receptor substrate p53 (IRSp53) and Calsenilin. The results of these two methods were then validated by real-time quantitative PCR. Both protocols exhibited identical mRNA expression patterns for IRSp53 and Calsenilin. In three brain regions analyzed, the levels of IRSp53 mRNA expression were ∼1.5-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure, although the relative abundance in regional expression levels was similar between the two methods. In contrast, the levels of Calsenilin mRNA expression were 10-17-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure and the relative abundance in regional expression levels was different. When compared to the real-time PCR results, the SQuISH™ method showed almost identical relative levels of IRSp53 to Calsenilin mRNA in all three brain regions analyzed, while the riboprobe-based procedure showed a completely opposite trend. These results support the accuracy of the SQuISH™ protocol for determining relative mRNA levels in the brain.

AB - In situ hybridization (ISH) is an essential technique for mapping gene expression in the brain. Although many ISH protocols provide for quantitative analysis of individual mRNAs in different brain regions or across experimental conditions, this technique has lacked the necessary standardization for quantitative comparisons between different mRNA transcripts. We have developed a standardized quantitative ISH (SQuISH™) protocol that utilizes multiple radioactive oligonucleotide probes, providing for increased sensitivity, decreased background and accurate comparison of relative mRNA levels. We evaluated the SQuISH™ protocol against a riboprobe-based ISH procedure by comparing the mRNA expression levels in the brain for two transcripts, insulin receptor substrate p53 (IRSp53) and Calsenilin. The results of these two methods were then validated by real-time quantitative PCR. Both protocols exhibited identical mRNA expression patterns for IRSp53 and Calsenilin. In three brain regions analyzed, the levels of IRSp53 mRNA expression were ∼1.5-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure, although the relative abundance in regional expression levels was similar between the two methods. In contrast, the levels of Calsenilin mRNA expression were 10-17-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure and the relative abundance in regional expression levels was different. When compared to the real-time PCR results, the SQuISH™ method showed almost identical relative levels of IRSp53 to Calsenilin mRNA in all three brain regions analyzed, while the riboprobe-based procedure showed a completely opposite trend. These results support the accuracy of the SQuISH™ protocol for determining relative mRNA levels in the brain.

KW - Calsenilin

KW - Insulin receptor substrate p53

KW - Neurotransmitters, modulators, transporters, and receptors

KW - Oligonucleotide probe

KW - Quantitative in situ hybridization

KW - Real-time quantitative PCR

KW - Signal transduction: gene expression

UR - http://www.scopus.com/inward/record.url?scp=1842505569&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1842505569&partnerID=8YFLogxK

U2 - 10.1016/j.brainres.2003.11.069

DO - 10.1016/j.brainres.2003.11.069

M3 - Article

C2 - 15053970

AN - SCOPUS:1842505569

VL - 1000

SP - 211

EP - 222

JO - Brain Research

JF - Brain Research

SN - 0006-8993

IS - 1-2

ER -