Use of bidirectional blots in differential display analysis

Daniel Kestler, Melissa Hill, Sujata Agarwal, Robert E. Hall

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-β, TNF-α, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA 32P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)216-220
Number of pages5
JournalAnalytical Biochemistry
Volume280
Issue number2
DOIs
StatePublished - May 1 2000
Externally publishedYes

Fingerprint

Complementary DNA
Display devices
Screening
Cytokines
Cloning
Endothelial cells
Human Umbilical Vein Endothelial Cells
Gene expression
Sepharose
Superoxide Dismutase
Organism Cloning
Interleukin-6
Plasmids
Clone Cells
Gels
Research Personnel
Modulation
Gene Expression

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Use of bidirectional blots in differential display analysis. / Kestler, Daniel; Hill, Melissa; Agarwal, Sujata; Hall, Robert E.

In: Analytical Biochemistry, Vol. 280, No. 2, 01.05.2000, p. 216-220.

Research output: Contribution to journalArticle

Kestler, Daniel ; Hill, Melissa ; Agarwal, Sujata ; Hall, Robert E. / Use of bidirectional blots in differential display analysis. In: Analytical Biochemistry. 2000 ; Vol. 280, No. 2. pp. 216-220.
@article{4293758dc88f4db4b90f96cbec2c95b8,
title = "Use of bidirectional blots in differential display analysis",
abstract = "We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-β, TNF-α, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA 32P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis. (C) 2000 Academic Press.",
author = "Daniel Kestler and Melissa Hill and Sujata Agarwal and Hall, {Robert E.}",
year = "2000",
month = "5",
day = "1",
doi = "10.1006/abio.2000.4532",
language = "English (US)",
volume = "280",
pages = "216--220",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Use of bidirectional blots in differential display analysis

AU - Kestler, Daniel

AU - Hill, Melissa

AU - Agarwal, Sujata

AU - Hall, Robert E.

PY - 2000/5/1

Y1 - 2000/5/1

N2 - We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-β, TNF-α, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA 32P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis. (C) 2000 Academic Press.

AB - We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-β, TNF-α, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA 32P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis. (C) 2000 Academic Press.

UR - http://www.scopus.com/inward/record.url?scp=0034193483&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034193483&partnerID=8YFLogxK

U2 - 10.1006/abio.2000.4532

DO - 10.1006/abio.2000.4532

M3 - Article

VL - 280

SP - 216

EP - 220

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -