[20] Analysis of altered gene expression by differential display

Peng, Liang; Bauer, David; Averboukh, Lidia; Warthoe, Peter; Rohrwild, Markus; Müller, Heiko; Strauß, Michael; Pardee, Arthur B.

doi:10.1016/0076-6879(95)54022-9

Cited by 124 publications

(72 citation statements)

References 12 publications

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“…Thus, in our study a total of about 21 000 cDNAs were ampli®ed and screened for changes in gene expression. Although not much is known about the intrinsic redundancy of this method, it is assumed that this number represents most of the mRNA-species expressed in a single cell (Liang and Pardee, 1995;Bauer et al, 1994). A total of 252 di erentially expressed bands were excised, of which 230 were successfully reampli®ed.…”

Section: Resultsmentioning

confidence: 99%

Identification of novel molecular markers which correlate with HPV-induced tumor progression

et al. 1998

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High risk HPVs (human papillomaviruses) are causally involved in the development of cervical cancer. However, other factors, such as somatic genetic alterations also play a decisive role in malignant conversion of cervical keratinocytes. Mutations and chromosomal aberrations are known to in¯uence the pattern of gene expression. Therefore we used the recently developed RT ± PCR based method of di erential display of mRNAs to detect di erences in gene expression which correlate with tumorigenicity. Non-tumorigenic HPV16-immortalized human foreskin keratinocytes (HPK IA) were compared with their tumorigenic counterparts and 49 di erent genes were identi®ed which were either up-or downregulated. The identi®ed genes encode proteins of the cytoskeleton and the extracellular matrix, the nuclear splicing apparatus, transcription regulators and membrane-associated proteins. The expression pattern of all genes was also examined by RNA ± RNA in situ hybridization in biopsies of normal cervical epithelium, precancerous lesions and cancers. Two genes, C4.8 and C21.7, are of particular interest because their expression is upregulated in a subset of high grade precancerous lesions and in over 58% of cancers. These two genes may therefore be considered as putative progression markers. C4.8 is a new member of the transmembrane 4 (TM-4) protein family which includes tumor-associated antigens such as CD63 and TAPA-1, whereas C21.7 shows no signi®cant homologies to any known genes or proteins.

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Section: Resultsmentioning

confidence: 99%

Identification of novel molecular markers which correlate with HPV-induced tumor progression

et al. 1998

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“…A number of genes potentially associated with tumor progression have been recently isolated by DD (Liang et al, 1995;Salesiotis et al, 1995;Francia et al, 1996;Wang et al, 1996). Figure 6 Time course of expression of 121A and Kif3c upon 2ME treatment.…”

Section: Discussionmentioning

confidence: 99%

Changes in gene expression during the growth arrest of HepG2 hepatoma cells induced by reducing agents or TGFβ1

Cabibbo¹,

Consalez²,

Sardella³

et al. 1998

Oncogene

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The growth of hepatoma cells can be inhibited by treatment with TGFb1 or with exogenous reducing agents. To gain information on the molecular mechanisms underlying growth arrest, we visualized and compared gene expression pro®les of proliferating versus non proliferating HepG2 cells by computer-assisted gene ®shing, an improved technique of RNA ®ngerprinting that allows the selective ampli®cation of coding regions within transcripts. While many transcripts are selectively regulated by either treatment, a set of bands appear to be coordinately regulated by 2ME and TGFb1, suggesting their possible involvement in the mechanisms of growth arrest. Display tags corresponding to 18 di erentially expressed genes were cloned and, in most cases, identi®ed as known genes or, more frequently, as their homospeci®c/cross-speci®c homologues. A novel member of the kinesin superfamily was identi®ed amongst the genes induced by both 2ME and TGFb1. This gene, KIF3C, is upregulated in several cell lines undergoing growth arrest. Taken together, our ®ndings show that computer-assisted gene ®shing is a powerful tool for the identi®cation and cloning of genes involved in the control of cell proliferation and indicate that extracellular reducing agents can regulate cell growth through modulation of gene expression.

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“…Differential display was performed with RNAimage Kit 1 mRNA differential display system from GenHunter (Nashville, TN, USA). 2 Each of the three reverse transcription reactions using T 11 G, T 11 A and T 11 C primers utilized 0.10 g of total RNA. Differentially expressed bands were excised, re-amplified, subcloned into pGEMT vector (Promega), and transformed into DH5␣ (Gibco-BRL Life Technology, Gaithersburg, MD, USA) according to standard procedure.…”

Section: Methodsmentioning

confidence: 99%

“…Reverse transcriptase then switches templates and extends to the end of the SMART oligonucleotide. First-strand cDNA corresponding to 20 ng of the starting RNA was PCR amplified with 1 × Advantage KlenTaq polymerase mix (1.1 g of KlenTaq-1 DNA polymerase and TaqStart Antibody; Clontech) using 0.2 M of dNTP and 1 M of amplification primer with sequence corresponding to the 5Ј end in both the CDS and SMART oligos (5Ј-AAGCAGTGGTATCAACGCAGAGT-3Ј) in 1 × KlenTaq PCR buffer (40 mM Tricine-KOH, 15 mM KOAc, 3.5 mM Mg (OAc) 2 , 75 g/mL BSA). Long-distance PCR was performed at 95°C for 15 s and 68°C for 5 min for 20 to 22 cycles (the optimal cycle number) in a Perkin Elmer DNA Thermal Cycler 480 (Perkin Elmer-Cetus, Norwalk, CT, USA).…”

Section: Methodsmentioning

confidence: 99%

A method for identifying differentially expressed genes in rare populations of primary human hematopoietic cells

Song²,

Gewirtz

1999

Leukemia

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Differential display (DD) is widely employed for identifying uniquely expressed genes within two different cell populations. While potentially powerful, DD is problematic because apparent positive clones require time-consuming verification which may be made even more difficult if only small amounts of starting material are available. We have devised a screening approach to address these issues in primary human hematopoietic cells. Candidate clones are identified in a slot-blot format and verified by 'Virtual Northern' blot analyses using globally amplified cDNA as the verification probe. This method is fast, and since it requires only ෂ0.2 g of total RNA, it is particularly useful when only limited amounts of study tissue are available.

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[20] Analysis of altered gene expression by differential display

Cited by 124 publications

References 12 publications

Identification of novel molecular markers which correlate with HPV-induced tumor progression

Identification of novel molecular markers which correlate with HPV-induced tumor progression

Changes in gene expression during the growth arrest of HepG2 hepatoma cells induced by reducing agents or TGFβ1

A method for identifying differentially expressed genes in rare populations of primary human hematopoietic cells

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