Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes

1993 ◽  
Vol 104 (4) ◽  
pp. 1187-1197 ◽  
Author(s):  
R.W. Dirks ◽  
F.M. van de Rijke ◽  
S. Fujishita ◽  
M. van der Ploeg ◽  
A.K. Raap
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Cultured Cells ◽  
Direct Detection ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Immediate Early ◽  
High Signal ◽  
Pretreatment Conditions

We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.

scholarly journals Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.

1995 ◽  
Vol 43 (10) ◽  
pp. 1005-1018 ◽  
Author(s):  
M V Macville ◽  
K C Wiesmeijer ◽  
R W Dirks ◽  
J A Fransen ◽  
A K Raap
Keyword(s):  
In Situ Hybridization ◽  
Cultured Cells ◽  
Elongation Factor ◽  
Housekeeping Gene ◽  
28S Rrna ◽  
Electron Microscopic ◽  
Rna Sequences ◽  
Gene Transcripts ◽  
Pre Treatment

We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde fixation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidase/diaminobenzidine (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxidase/DAB system. The accessibility of RNA in the different cell compartments was dependent on the extent of cross-linking during primary fixation even after permeabilization with saponin. By using the most optimal ISH protocol and the peroxidase/DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm but not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cytoplasm. The sensitivity of the method is such that HEF housekeeping gene transcripts were detected in the cytoplasm.

scholarly journals Detection of v- and c-erbB oncogene mRNA in cultured cells by in situ hybridization.

1990 ◽  
Vol 52 (1) ◽  
pp. 175-178
Author(s):  
Toshio IKEDA ◽  
Yasuhiro YOSHIKAWA ◽  
Kazuya YAMANOUCHI
Keyword(s):  
In Situ Hybridization ◽  
Cultured Cells

Detection of t(4;14)(p16.3;q32) Chromosomal Translocation in Multiple Myeloma by Double-Color Fluorescent In Situ Hybridization

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 724-732 ◽  
Author(s):  
Palma Finelli ◽  
Sonia Fabris ◽  
Savina Zagano ◽  
Luca Baldini ◽  
Daniela Intini ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
In Situ Hybridization ◽  
Cell Lines ◽  
Fluorescent In Situ Hybridization ◽  
Chromosomal Translocation ◽  
Fish Analysis ◽  
Interphase Nuclei ◽  
Igh Locus ◽  
Normal Controls

Abstract Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), variable chromosome partners have been identified by conventional cytogenetics, including the 11q13, 8q24, 18q21, and 6p21 loci. We and others have recently reported a novel, karyotypically undetectable chromosomal translocation t(4;14)(p16.3;q32) in MM-derived cell lines, as well as in primary tumors. The 4p16.3 breakpoints are relatively scattered and located less than 100 kb centromeric of the fibroblast growth factor receptor 3 (FGFR3) gene or within the recently identified WHSC1 gene, both of which are apparently deregulated by the translocation. To assess the frequency of the t(4;14)(p16.3;q32) translocation in MM, we performed a double-color fluorescent in situ hybridization (FISH) analysis of interphase nuclei with differently labeled probes specific for the IGH locus (a pool of plasmid clones specific for the IGH constant regions) or 4p16.3 (yeast artificial chromosome (YAC) 764-H1 spanning the region involved in breakpoints). Thirty MM patients, the MM-derived cell lines KMS-11 and OPM2, and six normal controls were examined. The identification of a t(4;14) translocation, evaluated as the presence of a der(14) chromosome, was based on the colocalization of signals specific for the two probes; a cutoff value of 15% (mean + 3 standard deviation [SD]) derived from the interphase FISH of the normal controls (range, 5% to 11%; mean ± SD, 8.16 ± 2.2) was used for the quantification analysis. In interphase FISH, five patients (one in clinical stage I, two in stage II, one in stage III, and a plasma cell leukemia) were found to be positive (≈15%). FISH metaphases with split or colocalized signals were detected in only two of the translocated cases and confirmed the pattern found in the interphase nuclei. Furthermore, in three of the five cases with the translocation, FISH analysis with the IGH joining probe (JH) showed the presence of the reciprocal product of the translocation [der(4) chromosome]. Overall, our study indicates that the t(4;14)(p16.3;q32) chromosomal translocation is a recurrent event in MM tumors and may contribute towards the detection of this lesion and our understanding of its pathogenetic and clinical implications in MM.

scholarly journals Comparison of 35S and chemiluminescence for HPV in situ hybridization in carcinoma cell lines and on human cervical intraepithelial neoplasia.

1996 ◽  
Vol 44 (7) ◽  
pp. 665-671 ◽  
Author(s):  
P Lorimier ◽  
L Lamarcq ◽  
A Negoescu ◽  
C Robert ◽  
F Labat-Moleur ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Ccd Camera ◽  
Intraepithelial Neoplasia ◽  
Hpv 16 ◽  
Human Carcinoma ◽  
Carcinoma Cell Lines ◽  
Biopsy Specimens

For in situ hybridization (ISH), development of sensitive, nontoxic alternatives to the use of radioactivity is a constant concern. In this trend, and close to chromogenes and fluorophores, chemiluminescence appears an attractive method. A first positive experience in immunocytochemistry and in ISH, by using the enhanced luminol as luminogene substrate for horseradish peroxidase (HRP) led us to compare the sensitivity of 35S autoradiography and chemiluminescence. For this purpose, we used three human carcinoma cell lines, CaSki [400-600 copies of human papilloma virus (HPV) 16], HeLa (10-50 copies of HPV 18), and SiHa (1-5 copies of HPV 16), and 40 biopsy specimens of human cervical preneoplastic and neoplastic lesions. We performed ISH by using HPV cDNA biotin-labeled probes, detected by a two-step immunocytochemical reaction, the secondary antibodies being either 35S-labeled for autoradiography or HRP-labeled for chemiluminescence. An intensified CCD camera allowed acquisition of the luminescent signal. After only 10 min of photon accumulation, on cell line smears as well as on serial tissue sections, chemiluminescence gave comparable results to those obtained by a 3-week exposure for 35S autoradiography. A quantitative approach on cervical biopsy specimens confirmed this similar level of sensitivity by measuring the area of 35S- or chemiluminescence-stained nuclei. Our results indicate that chemiluminescence is a credible and perfectible alternative to radioisotopes for in situ detection of nucleic acids by hybridization.

scholarly journals A New Subtype of Pre-B Acute Lymphoblastic Leukemia With t(5; 12)(q31q33; p12), Molecularly and Cytogenetically Distinct From t(5; 12) in Chronic Myelomonocytic Leukemia

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1716-1722 ◽  
Author(s):  
Iwona Wlodarska ◽  
Anna Aventı́n ◽  
Júlia Inglés-Esteve ◽  
Daniela Falzetti ◽  
Arnold Criel ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Acute Lymphoblastic Leukemia ◽  
Fluorescence In Situ Hybridization ◽  
Cell Lines ◽  
Chromosomal Abnormality ◽  
Gene Fusion ◽  
Lymphoblastic Leukemia ◽  
Chronic Myelomonocytic Leukemia ◽  
Myelomonocytic Leukemia

Abstract Translocation t(5; 12)(q33; p13), resulting in an ETV6/PDGFRB gene fusion, is a recurrent chromosomal abnormality associated with chronic myelomonocytic leukemia (CMML). An analogous translocation was also found in four cell lines with features of pre-B acute lymphoblastic leukemia (ALL). Using fluorescence in situ hybridization (FISH) we show here that in three of these cell lines identical complex rearrangements occurred. However, the regions involved on 5q and 12p are different from the breakpoints in CMML, and the translocation is accompanied by seemingly identical cryptic deletions of both 5q and 12p chromosome sequences in all analyzed pre-B ALL cell lines. The similar cytogenetic, FISH, and immunophenotyping findings in the three cell lines suggest that the t(5; 12)(q31q33; p12) defines a new entity of pre-B ALL.

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