An improved procedure for in situ RTPCR

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Isolation and Quantification of Axonal mRNAs Using Porous Membrane Inserts and RTddPCR
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L Volpi1, L Doneda, M G Grimoldi

  • 1Dept. of Biology and Genetics, University of Milan, Milan, Italy.

Molecular and Cellular Probes
|February 1, 1996
PubMed

Abstract:

Direct in situ RTPCR has been successfully applied to paraffin-embedded human placental specimens. The expression of stem cell factor (SCF) mRNA visualized in cytotrophoblast and stromal cells is validated by the lack of any specific signal in parallel specimens where reverse transcription is omitted.

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
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