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- W1593095412 abstract "This chapter describes microadapted techniques to isolate messenger RNA (mRNA), or total RNA from only a single cell. The isolated mRNA is directly processed for complementary DNA polymerase chain reactions (cDNA PCR), whereas the total RNA is treated with DNase I to remove contaminating genomic DNA. The DNase I-treated total RNA is reverse-transcribed into cDNA and then used for cDNA-PCR analysis. To determine the mRNA phenotype of cells by cDNA-PCR, two oligonucleotide primers that can recognize the (+)-strand and the (–)-strand of the target gene are required. The target sequence is amplified exponentially by repeated cycles of denaturation, primer annealing, and primer extension in the presence of thermostable DNA polymerase. The analysis of the mRNA phenotype of a small number of cells by cDNA-PCR requires the isolation of sufficient quantities of RNA. This approach is hampered by the fact that standard protocols for the isolation of RNA cannot be applied. Therefore, techniques scaled down for the isolation of small amounts of RNA, such as the microadapted guanidine thiocyanate (GuSCN)/CsCl gradient centrifugation, and protocols for the direct isolation of poly(A) RNA from cellular lysates have been developed." @default.
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- W1593095412 date "1995-01-01" @default.
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- W1593095412 title "[5] Single-cell cDNA-PCR" @default.
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- W1593095412 doi "https://doi.org/10.1016/s1043-9471(06)80083-2" @default.
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