CYTOCHEMICAL HYBRIDIZATION WITH FLUOROCHROME-LABELED RNA .2. APPLICATIONS
CYTOCHEMICAL HYBRIDIZATION WITH FLUOROCHROME-LABELED RNA .2. APPLICATIONS
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DOI：
10.1177/29.2.6166654
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发表时间：
1981-01-01
影响因子：
3.2
通讯作者：
VANDUIJN, P
中科院分区：
文献类型：
--
作者：
BAUMAN, JGJ;WIEGANT, J;VANDUIJN, P
用荧光标记的RNA与细胞化学杂交检测特定DNA序列，并在荧光显微镜下检测杂交体。用四甲基罗丹明异硫氰酸肼衍生物(TRITC)标记与昆虫锥虫动泡体DNA或腺病毒5型DNA互补的RNA。通过DNA与琼脂糖珠的交叉杂交实验，研究了~3H标记的互补RNA与四甲基罗丹明反应的特异性。反应的程度通过珠悬浮液的闪烁计数和单个琼脂糖珠的定量荧光显微镜来测量。证明了罗丹明标记的cRNA具有杂交的能力，并且荧光标记没有干扰杂交反应的特异性。对光肩星天牛细胞进行细胞化学杂交后，罗丹明标记的动泡体cRNA仅对动泡体进行染色。在细胞核内未观察到荧光。用罗丹明标记的Ad-5cRNA与病毒感染的人口腔上皮样癌KB细胞进行细胞化学杂交后，在细胞核内观察到明显的染色模式。在未感染的细胞中或与异源罗丹明标记的RNA杂交后，未见荧光。讨论了与罗丹明标记的RNA进行细胞化学杂交的可能性和局限性。
The cytochemical detection of specific DNA sequences by hybridization with fluorochrome-labeled RNA and detection of the hybrids by fluorescence microscopy is described. RNA complementary to the DNA of the kinetoplasts of Crithidia luciliae (an insect trypanosome) or to adenovirus-5 (Ad-5) DNA were labeled with the hydrazine derivative of tetramethylrhodamine isothiocyanate (TRITC). The specificity of the reactions between the complementary RNA labeled with 3H and tetramethylrhodamine was studied by cross-hybridization experiments using a model system in which the DNA were bound to Sepharose beads. The extent of the reaction was measured by scintillation counting of the bead suspensions and quantitative fluorescence microscopy of individual Sepharose beads. The ability of the rhodamine-labeled cRNA to hybridize and the absence of interference of the fluorochrome label with the specificity of the hybridization reaction was demonstrated. After cytochemical hybridization on microscopic preparations of C. luciliae cells, the rhodamine-labeled kinetoplast cRNA stains only the kinetoplasts. No fluorescence was observed in the nuclei. After cytochemical hybridization of rhodamine-labeled Ad-5 cRNA with virus infected KB [human male oral epidermoid carcinoma] cells, a distinct staining pattern in the nuclei was observed. No fluorescence was seen in uninfected cells or after hybridization with heterologous rhodamine-labeled RNA. The possibilities and limitations of cytochemical hybridization with rhodamine-labeled RNA are discussed.