Reverse Transcriptase In Situ PCR

Reverse Transcriptase In Situ PCR.

Reverse Transcriptase In Situ PCR New Methods in Cellular Interrogation

With the introduction of the technique, reverse transcriptase polymerase chain reaction (RT-PCR), marks a leap in sensitivity over many other methods of detecting mRNA transcripts, such as Northern blotting.  The introduction of sensitive techniques such as RT-PCR, it has become possible to amplify RNA transcripts from very small amounts of template nucleic acid, thus opening new avenues of research that were previously off limits because of difficulties in obtaining adequate quantities and quality of RNA.  Even though all the benefits that RT-PCR possesses there are some downfalls it has as well. RT-PCR has the same limitations as its predecessor because the isolation of RNA necessitates the destruction of the cells/tissue involved, therefore not allowing the identification of the specific cell source of the mRNA.  On the other hand, in situ hybridization gives the specific localization of mRNA to the cells of origin, but the methodology is much less sensitive than RT-PCR.  A technique that incorporates the best attributes of in situ hybridization (specific cellular localization) and RT-PCR (high sensitivity) would be ideal.

RT in situ PCR provides these attributes, allowing for the location and detection of low copy RNA species, amplified within individual intact cells.  The method of RT in situ PCR involves fixation of cells in suspension, followed by controlled digestion of the crosslinked cellular proteins with a proteolytic enzyme.  This technique allows the entry of the RT and PCR reagents into the cell.  Next, genomic DNA is removed by a Dnase digestion step, thus ensuring that only mRNA is amplified.  Subsequent steps of RT and PCR are then undertaken, using a “labeled” reporter nucleotide, which results in its direct incorporation into the PCR product.  This is followed by a detection step that visualizes the amplified mRNA, either by chromogenic or radioactive means.  The specificity of the reaction can be then established by several methods.  Methods of RT in situ PCR, although sharing fundamental steps, have varied greatly between laboratories.  Based on experience, RT in situ PCR seems to be best suited for the detection of mRNA in single-cell suspensions, in which fixation and pretreatments can be optimally controlled.  The method outlined below has been built upon through our experiences.  The inclusion of appropriate controls in every run helps insure accurate results.  Controls to indicate that genomic DNA has been removed as a source of false-positive signals (negative control), or failure of PCR amplification because of inadequate protease digestion or flaws in the RT-PCR protocol resulting in false negative results (positive control) must be tested on every slide.  Therefore, a slide schematic of the necessary controls and test spots is included to aid in the elimination of spurious results.

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