In situ localization of c-myc mRNA in HL-60 cells using non-radioactive synthetic oligodeoxynucleotide probes.

Nakane

1990

Self Cite

To analyze the expression of specific mRNA at the level of individual cells, non-radioactive in situ hybridization has been a most powerful technique. In the process of in situ hybridization, the use of formamide is usually required in order to reduce the melting temperature (Tm) of nucleic acids. However, formamide is an expensive and unstable reagent, and more importantly, formamide in itself has some deteriorative effects such as nonspecific staining and morphological damage on the results of in situ hybridization. In this study, we examined the use of a mixture (Nm) of nucleotides (AMP, GMP, UMP, CMP) as an alternative to formamide in our non-radioactive hybridization system with thymine-thymine (T-T) dimerized DNA probe. When the effects of Nm on re-annealing of denatured pBR 322 DNA were investigated by electrophoretic patterns on agarose gel, it was confirmed that Nm (about 20-200 mg/ml) reduced the Tm of pBR 322 DNA. On dotblot hybridization using Nm, we obtained sensitive and specific results similar to that of formamide. Finally, on frozen sections of rat pituitary glands fixed by perfusion of 4% paraformaldehyde in phosphate buffered saline (pH 7.4), prolactin mRNA was successfully localized in situ using Nm instead of formamide.Non-radioactive in situ hybridization has been a most effective method for localization of specific mRNA in individual cells (4,6,19,20). Recently we have developed a new method (10, 11, 17) using thymine-thymine (T-T) dimerized DNA as a nonradioactive probe, which is thought to be the simplest in terms of no requirements for the separation of labeled DNA from unreacted labeling compounds, and we have further shown a wide applicability of the method (7,8,9,18,21). In parallel with the approach, using T-T dimerized DNA probes we have tried to establish a tissue processing procedure best suited to non-radioactive in situ hybridization (10, 16).In molecular hybridization, in order to obtain specific hybrids between probe nucleic acids and target nucleic acids, hybridization reaction should be carrid out near the melting temperature (Tm) of the corresponding double-stranded nucleic acids, theoretically. In practice, however, it is recommended that hybridization be carried out at a temperature of (Tm-25)°C (for filter hybridization) or (Tm-30)°C (for in situ hybridization) (3). In most of the cases to hybridize between probe DNA and target mRNA, Tm-30 was calculated to be higher than 65-75°C (22), while to preserve the morphology of specimens hybridization should be carried out at a temperature lower than 45°C. Since it is thought to be prerequisite to preserve the morphology of cells and tissues for a full implementation of in situ hybridization, the use of some reagents

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Use of nucleotides as an alternative to formamide in non-radioactive in situ hybridization.

Nakane

1990

Self Cite

show abstract

“…Immunohistochemical detection of T-T dimerized DNA, which was fixed onto a nitrocellulose filter, was described in the previous paper [10,14]. Briefly, a 2 ,ulaliquot of T-T dimerized A phage DNA solution, which was irradiated with ultraviolet-light at 10 KJ/m2, was applied onto a 20 X SSC (SSC; 0.15 M NaCI and 0.015 M sodium citrate, pH 7.0) treated nitrocellulose filter at 0.1 pg -10 ng per spot.…”

Section: Immunohistochemical Detection Of T -T Dimerized Dnamentioning

confidence: 99%

“…Recent advances in this field have included introduction of various haptens such as digoxigenin [6,11,19] and thymine-thymine (T-T) dimer [10,12,13] to label probe nucleic acids, use of synthetic oligonucleotides as probe nucleic acids [8,14,16], and development of an evaluation system of hybridizable RNA retention using 28S rRNA as reference [7,22,23]. The resultant improvements in the protocol of nonradioactive in situ hybridization lead to a greater detection sensitivity.…”

Section: Introductionmentioning

confidence: 99%

A Novel Amplification Method of Nonradioactive In Situ Hybridization Signal for Specific RNA with Biotinylated Tyramine.

Kanemitsu

Hoshino

et al. 1997

Self Cite

“…Thymine-thymine was introduced at the 5' and 3' ends to obtain a thymine-thymine dimerized DNA probe after UV irradiation [16,25]. The oligonucleotide used as a probe for the detection of H. pylori was derived from the sequenced urease gene and did not cross-hybridize with urease genes from other species theoretically [4].…”

Section: Dna Probesmentioning

confidence: 99%

Detection of a Helicobacter pylori Gene Marker in Gastric Biopsy Samples by Non-radioactive in situ Hybridization.

Sugawara

Onodera

et al. 1995

Self Cite