Direct in situ reverse transcriptase-polymerase chain reaction.
Direct in situ reverse transcriptase-polymerase chain reaction.
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直接原位逆转录酶-聚合酶链反应。
DOI：
10.1152/ajpcell.2001.281.2.c726
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Bacallao,R
中科院分区：
文献类型：
--
作者：
Kher,R;Bacallao,R
In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.
