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More on the in situ miRNA detection methods...
In situ detection of miRNA expression poses several technical challenges that are addressed in Phylogeny’s services. Foremost, methods are needed that can be used with paraffin embedded formalin-fixed tissues so as to benefit from the wealth of immunohistochemical analyses that can be done on such samples in parallel. Cell and tissue fixation methods have a significant impact on the success of in situ techniques and miRNA methods are no different. Phylogeny’s expertise and experience with in situ methods is thus an invaluable asset for miRNA research.
RT-in situ PCR
Reverse Transcriptase (RT) -in situ PCR uses a PCR reaction to amplify a target which can either be detected by direct labelling during the amplification or by subsequent hybridization with a labelled probe. For RT-in situ PCR a critical variable is the complete removal of genomic DNA which can otherwise act as a non specific primer for PCR reactions. It is critical that the optimal protease digestion time for a given tissue/cell preparation should be determined. Primers that are adequate for solution phase RT PCR will also support RT in situ PCR, including for any miRNA. Primers for the miR precursors have been designed to anneal primarily to the stem portion of the precursor hairpin.
In situ hybridization with chemically modified LNA probes
The key variable for the success of in situ hybridization is probe size (which affects the melting temperature( Tm) of the probe/target complex). Probes can be chemically synthesized or prepared by nick translation of a larger fragment. The melting temperature™ of cDNA-miRNA complexes are low and with most standard in situ hybridization cocktails, is near room temperature, making it difficult to distinguish a signal from background. This problem can be remedied by modifying some of the nucleotide bases using a locked nucleic acids (LNA) modification in which altered nucleotides becomes more rigid in 3-D space. The LNA modified nucleotides substantially increase the Tm of the probe/target complex, yielding a better signal to noise ratio.
Interpretation of results and overlap with other methods
Good negative and positive controls are essential when doing in situ hybridization in general and for miRNAs in particular.
The key points to realize with RT in situ PCR microRNA detection is that it can be used to detect the precursor molecule, and that a negative result effectively rules out the presence of the precursor in a given cell. This is in contrast to in situ hybridization with an LNA probe, where a negative result does not rule out the presence of either the precursor or mature form of a given miRNA due to the relatively lower sensitivity of Northern blotting relative to PCR.
By using RT in situ PCR, one can ascertain whether the precursor of a given miR is being produced in the tissue of interest. By combining the results with in situ hybridization with the LNA probe on serial sections, one can study the same cells and determine whether the precursor is being robustly processed to the mature form; a positive in situ result would confirm this, whereas a negative result with the LNA probe (assuming the controls worked properly) would strongly suggest that the precursor is not being actively processed.
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