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At least 289 records · Page 16Linked to original sources
PubMed1991

[A simple and sensitive DNA-RNA hybridization method--in situ hybridization on the nitrocellulose filter for detecting oncogene expression in tumor cells by using digoxigenin-labelled probes].

A simple and sensitive method for detecting oncogene mRNA is performed by fixing tumor cells directly onto nitrocellulose paper, followed by in situ RNA hybridization with digoxigenin-labelled oncogene probes. The human lung large cell carcinoma cell line PLA801-D95 (before and after induction of differentiation with retinoid acid) and leukemic cells from clinical patients were used as target cells. The results showed that 5000 cells were sufficient to give a positive signal for oncogene mRNA. Compared with RNA dot blot, it was found that this technique was quick, simple, sensitive and reproducible. It can be used to study gene expression in tumor cells as well as other cells.

Carcinoma↗
PubMed1989

[An alpha-satellite DNA sequence, alpha-RI-6, specific for human chromosomes 13 and 21, detected using the RFLP technic with digoxigenin labelled probes].

The authors compared two at present most widely used techniques for labelling DNA probes: a) radioactive labelling by means of the radioisotope 32P; non-radioactive labelling using the hapten digoxigenin for the visualization of the hybridization process on nylon membranes. Then sensitivity of the technique of non-radioactive labelling of heterochromatin probes was equivalent to the radioactive method.

Blotting, Southern↗
PubMed1989

[Methodologic variants of DNA blood stain analysis: extraction in agarose and digoxigenin marking].

DNA was extracted from 1 microliter bloodstains on glass, cotton cloth and paper using proteinase K digestion in a solution containing low gelling temperature agarose. This method is advantageous for small amounts of source material because the purifying procedures of other methods that contain a risk of loss of DNA, are avoided. The recovered DNA was digested with Hae III and after Southern blotting, a Digoxigenin labeled probe (Amprobe, human Y-chromosome specific repeat) was successfully used for sex determination. With this non-radioactive method quantities of DNA smaller than 1.5 pg were detected.

Blood Grouping and Crossmatching↗
PubMedDate not supplied

In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells.

To establish a nonradioactive method for demonstrating HPV DNA in routinely treated smears of the uterine cervix (alcohol fixation, staining according to Papanicolaou, preservation), in situ hybridizations were carried out in HeLa and SiHa cells grown on slides. After detailed investigations, the sensitivity and specificity of the biotin-avidin method (10) initially used proved to be inadequate for this purpose. Demonstration of HPV 16 DNA in SiHa cells (SiHa cells only contain 1-2 HPV genome copies) was possible only by use of digoxigenin-labeled HPV 16 gene probes, as well as an improved purification of the sample DNA from vector contaminations. Thus, for the first time a protocol for correlation of the results of an in situ hybridization with the cytological appraisal in the very same smear preparation has been developed for routine diagnostics.

Biotechnology↗
PubMed1980

Metabolism and rate of elimination of digoxigenin bisdigitoxoside in dogs before and during chronic azotemia.

The purpose of this study was to evaluate the metabolism and rate of elimination of digoxigenin bisdigitoxoside (bis) before and during chronic azotemia in dogs. Bis was eliminated primarily by nonrenal mechanisms. The half-life of bis was 18.5 hr, compared to 31.6 hr for digoxin, and was not significantly increased in azotemic dogs. The oral bioavailability of bis in azotemic dogs relative to an intravenous dose was approximately 46%.

Animals↗
PubMed1995

Noise-free chemiluminescent detection of human T cell receptor and interleukin-2 receptor genes after optimization of digoxigenin labeled probe concentration.

Reduction in the background problems to improve the efficiency of nonradioactive labeling and detection procedure has been the focus of attention in the recent past for wider acceptance of the technique in nucleic acid research. We have achieved success in obtaining a relatively background free detection of single copy genes such as, T-cell receptor-delta (TCR-delta) and interleukin-2 receptor (L-2r) in human genome by using optimized concentrations of the digoxigenin labeled probes. Inclusion of such an optimization step for each probe before carrying out the actual hybridization experiment did not require any further modification in hybridization conditions and detection protocols as suggested earlier [Anal Biochem, 210 (1993) 235; Colloq Boehringer Mannheim, 2 (1991) 4.]

Blotting, Southern↗
PubMed1993

Expression of alpha 2,6-sialylated sugar chains in normal and neoplastic colon tissues. Detection by digoxigenin-conjugated Sambucus nigra agglutinin.

It has previously been reported that about 90% of human colon carcinomas express increased levels of the sialyltransferase which adds sialic acid in alpha 2,6 linkage to galactose residues on N-linked chains of glycoproteins. To ascertain whether colon cancer tissues actually express increased amounts of alpha 2,6-sialylated sugar chains on their glycoconjugates, we screened tissue sections of normal colon, benign and malignant colon tumors with digoxigenin-conjugated Sambucus nigra agglutinin (SNA), a NeuAc alpha 2,6Gal/GalNAc-specific lectin. At the concentration of lectin used, epithelial cells of all the 13 normal colon specimens examined were unreactive; 3 out of 8 benign lesions showed a weak reactivity being the remainder unreactive, while 23 out of 26 carcinomas were positive at a variable degree. Qualitative differences were evident among different carcinoma specimens. In some cases a large number of intensely stained intracytoplasmatic particles was present, thus suggesting that reactivity may be associated with secretions, very likely of mucus droplets. In other specimens there was a more uniform distribution of the staining which suggest that reactivity is associated with cell membrane glycoconjugates. These data indicate that the expression of a2,6-sialylated sugar chains is remarkably increased in the majority of colon cancer specimens examined.

Adenoma↗
PubMed1995

Random primer p(dN)6-digoxigenin labeling for quantitation of mRNA by Q-RT-PCR and ELISA.

The ability to accurately measure mRNA levels in samples of total RNA is essential for studies on control of gene expression. The mRNAs from the housekeeping gene for phosphoglycerate kinase (PGK-1) can serve as a quality control for RNA samples. We describe an enzyme-linked immunosorbent assay (ELISA) method for mRNA determination by Q-RT-PCR, a quantitative reverse transcriptase-mediated PCR assay with competitive internal standards. After PCR, two biotinylated capture primers, one specific for PGK-1 cDNA and another one for internal standard, are annealed in separate assays so that each can attach DNA to a streptavidin-coated microplate. The captured DNA is either internally labeled with digoxigenin (DIG) or is "developed" after annealing with DIG-labeled primers. Bound DNA is then quantitated by adding DIG-specific antibody with attached alkaline phosphatase and measuring phosphatase activity with a chromogenic substrate and a plate reader. We compared different capturing methods and various primers labeled with DIG at their 3' ends. We determined that amplified PGK-1 DNA specifically captured with biotinylated primers was efficiently assayed with random p(dN)6-DIG.

Base Sequence↗
PubMed1995

Resin-purified digoxigenin-labeled DNA probes: an efficient protocol for mapping and fingerprinting sugarcane.

Nonradioactive Southern blotting using digoxigenin (DIG) has become routinely applied to the analysis of single-copy genes for genetic mapping because it is fast and safe. Previous studies indicate that DIG-labeled probes are suitable for single-gene detection in less complex genomes, but their efficient application to mapping a large octoploid genome has not been discussed. We developed a stream-lined procedure for nonradioactive restriction fragment length polymorphism mapping and DNA fingerprinting of sugarcane that combines DIG-11-dUTP and anion-exchange chromatography. In this report, we show that anion-exchange chromatography provides a reliable and simple technique for the resin-purification of large numbers of DIG-labeled DNA fragments 0.3-3.0 kb in size, and it is essential in minimizing contaminants and nonspecific signal.

Anion Exchange Resins↗
PubMed1995

In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.

We have developed an easy, stream-lined yet sensitive protocol for in situ hybridization to mRNA in frozen tissue sections or cytospins using digoxigenin-labeled RNA probes detected by alkaline phosphatase. We found the crucial parameters for successfully performing this technique to be tissue quality, fixation time and effective removal of excess probe. Most preparation, incubation steps and washes as described in the literature were found to be unnecessary and, therefore, eliminated, making this protocol simple enough to be achieved in any laboratory with access to tissue preparation and some molecular biology expertise.

Alkaline Phosphatase↗
PubMed1994

Digoxigenin-labelled molecular probe for the simultaneous detection of three potato pathogens: potato spindle tuber viroid (PSTVd), potato virus Y (PVY), and potato leafroll virus (PLRV).

A molecular probe, p3POT, was constructed of PSTVd, PVY, PLRV cDNA fragments introduced into pUC18 vector. Sequencing of the inserts revealed that cloned fragments covered conservative parts of pathogenic genomes. Dot-blot hybridization of digoxigenin-labelled construct to crude extracts from plants infected with different potato viruses proved high sensitivity and specificity of the p3POT probe. This makes p3POT probe an useful tool for the routine testing, and selection of virus-free potatoes.

DNA Probes↗
PubMed1995

[Detection of distemper virus N protein RNA in the brain of dogs with spontaneous distemper encephalitis using a digoxigenin-labeled, double-stranded DNA probe for in situ hybridization].

A digoxigenin-labelled dsDNA-probe of 287 basepairs length complementary to the nucleoprotein-gene of canine distemper virus (CDV) was generated by the polymerase-chain-reaction. The dsDNA-probe hybridized specifically with base sequences of 8 different CDV strains, whereas no hybridization was observed with a porpoise and a canine parainfluenza virus and only a weak signal was obtained with measles virus. In formalin-fixed, paraffin-embedded brain sections of 35 immunohistologically CDV antigen positive dogs with spontaneous distemper encephalitis CDV-RNA could be detected in 25 cases by in situ hybridization. The reason for the lack of RNA detection in some immunohistologically positive dogs may be due to the low stability of DNA-RNA-hybrids. Degradation of RNA by RNAses or diffusion out of autolysed cells can not be excluded.

Animals↗
PubMed1994

[Preparation of digoxigenin labelled probe and detection of HBV DNA in liver and extrahepatic tissue with in situ hybridization].

A new rapid technique for intrahepatic and extrahepatic HBV DNA detection by using digoxigenin (dig) labelled probe with in situ hybridization was developed. This technique has the advantage of being non-radioactive and a quick procedure yielding stable results and showing a clear background. 45 liver specimens were tested with this technique. Among the patients with positive intrahepatic HBsAg and HBcAg, positive detection of HBV DNA was highest (77.27%, 17/22). Some results were confirmed by PCR test. 19 extrahepatic specimens were detected with in situ hybridization. HBV DNA was seen clearly in the nuclei of myocardial cells, pancreatic islet cell, renal tubule epithelial cells and testicular spermatogenic cells. The results of this study might contribute to the study of molecular mechanism of HBV-induced injury in liver cells and extrahepatic tissue.

Base Sequence↗
PubMed1994

Localization of ZP3 mRNA in mouse ovary by non-radioactive in situ hybridization with digoxigenin-labelled cDNA.

In situ hybridization using digoxigenin-labelled cDNA has dual advantages of having high structural resolution and high sensitivity and avoiding problems associated with the handling of radioactive materials. We have used this technique to detect and locate mRNA for mouse ZP3 protein in paraffin sections of fixed ovaries of newborn and adult mice. Mouse ZP3 mRNA was restricted to the cytoplasm of oocytes. After day 16, ZP3 mRNA expression was increasingly restricted to the cytoplasm of the oocyte. Previous work on the distribution of ZP3 mRNA was confirmed and we were able to show that using non-isotopic in situ hybridization, sensitive and accurate cellular localization was feasible in paraffin embedded mouse ovary.

Alkaline Phosphatase↗
PubMed1993

Quantitative and sensitive northern blot hybridization using PCR-generated DNA probes labeled with digoxigenin by nick translation.

Northern blot hybridization is one of the most convenient methods of detecting an mRNA. Nonradioactive Northern blotting using digoxigenin (DIG) is becoming widely applied because it is rapid and safe. Previous studies have indicated that DIG-labeled RNA probes are suitable for Northern blot hybridization. Here, the application of PCR-generated double-stranded DNA probes labeled with DIG by nick translation is described. DNA probes were synthesized by PCR, then labeled with DIG by nick translation. Northern blot hybridization was performed using the DIG-labeled DNA probes, and the signals were detected by means of a chemiluminescent reaction. A low amount of DIG-dUTP in the labeling reaction resulted in excellent Northern blots with low background. Densitometric analysis of the blots showed that the mRNA concentrations could be determined by densitometric analysis. The sensitivity of the DIG-Northern system was comparable to Northern blotting using 32P and was sufficiently sensitive to detect low-abundance mRNA.

Animals↗
PubMed1994

Localization of mRNA of procollagen alpha 1 type I in torn supraspinatus tendons. In situ hybridization using digoxigenin labeled oligonucleotide probe.

A tendon is predominantly composed of collagen Type I. To determine the synthesis of collagen Type I after a rotator cuff tear, an in situ hybridization technique was applied to localize cells containing procollagen alpha 1 Type I in the proximal stump of five torn supraspinatus tendons obtained during surgery. Specimens were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 6 microns. A 22 mer oligonucleotide corresponding to a sequence coding a part of human procollagen alpha 1 Type I messenger RNA (mRNA) was used as a hybridization probe. The probe was 3'-end labeled with digoxigenin-11-dUTP, and the probe-mRNA hybrids were enzymatically visualized using conventional chromogens for alkaline phosphatase. The procollagen alpha 1 type I mRNA was clearly observed in the cells near the margin of the tear. However, they were not consistently found in the vicinity of the intratendinous extension of the tear and in cells of the subacromial bursa. It is concluded that this method should be used to study the characteristics of collagen synthesis in a torn rotator cuff tendon.

Digoxigenin↗
PubMed1993

[Detection of human papilloma virus DNA in tissue sections of cervical carcinoma by in situ hybridization with digoxigenin labelled DNA probes].

Formalin fixed, paraffin embedded tissues from 48 cases of squamous carcinoma of the cervix were examined for the presence of human papilloma virus (HPV) DNA by in situ hybridization using digoxigenin labelled HPV 6, 11, 16 and 18 DNA probes respectively. HPV DNA was found in 23 of the tumors (47.92%). HPV 16 DNA in 21 tumors (43.75%), HPV 18 DNA in four tumors (8.33%), no tumors hybridized to either HPV 6 DNA or HPV 11 DNA. HPV DNA was mainly observed in the nuclei of koilocytic cells and carcinoma cells, partly in the cytoplasm of carcinoma cells. These results show that the presence and typing of HPV in the cervix uteri may be studied by in situ hybridization and confirm the close association between cervical cancer and HPV (especially HPV 16 and 18) infection.

Carcinoma, Squamous Cell↗
PubMed1993

[Preparation of digoxigenin labeled virG DNA probe of Shigella and its homology analysis].

Using hapten digoxigenin labeled 1. 4kb virG DNA probe of S. flexneri 2a YSH6000. We examined the DNA homology among 73 strains of Shigella, 32 strains of EIEC, 17 strains of Yersinia, 5 strains of S. typhi and 5 strains of E. coli. At the same time strains of Shigella, EIEC and Yesinia were tested by Sereny assay. We concluded that virG DNA is conserved both in Shigella and EIEC and is related to Sereny test only in Shigella and EIEC but not in Yesinia. The coincidence rate of hybridization and Sereny test was 87.5% and 90.9% for S. sonni and EIEC respectively.

Bacterial Proteins↗