In situ PCR harnesses the power of #PCR or #RTPCR and #Histology to answer specific and complex #diagnostic questions in a fairly unimaginable (at least to me!) way! I mean who would have thought dead cells will allow #DNApolymerase to amplify its DNA!
Principle
The essence of in situ PCR is the use of cells as PCR tubes. Here the PCR reagents enter cells, finds specific regions of DNA, amplify them and through signal readout systems, inform the user different cellular information. The protocol is pretty critical and is more focused on #immunohistochemistry than #molecularbiology.
Protocol
Sample fixation: This includes fixation of the tissue into its native form and morphology with 2-4% #Paraformaldehyde or 2-3% #Glutaraldehyde solution.
Sample sectioning and de-paraffinization: Then the tissue is sectioned with a #microtome according to the thickness needed. It is then heated at 80C to melt the paraffin and cycled through Xylene and Ethanol to de-paraffinize.
Slide preparation: The glass slides can be treated with 0.1M #Triethanolamine and 0.25% #Aceticanhydride to reduce the static charge on them.
Sample permeabilization: Upon placing the thin slices of tissue on slides, the sample is treated with #ProteinaseK or #Pepsin to form pores through the cell membrane, so that the #PCR reagents like polymerases can access the DNA/ RNA.
PCR/ RT PCR: PCR/ RT PCR reactions are pretty straightforward here with only specialities being the use of #PCRstabilizers like #Gelatin and #BSA and substituting the regular 0.2mM final concentration of #dNTPs with 4uM final concentration of DIG-11-dUTPs. 10-15uL of the PCR mix cautiously pipetted into the well of the slide with the tissue and placed in appropriate wells of a thermal cycler. In most protocols, initial denaturation and final extension parts are omitted from the cycle, although some protocols have retained them.
Signal detection: PCR with #DIG-11-#dUTPs generates products with DIG labels. After PCR, the cells are dipped into 1:500 diluted Anti-DIG-#AlkalinePhosphatase (AP) antibody. Consequently #BMPurple or any other AP substrate is added to the wells, incubated and viewed under #BrightField or #Fluorescence microscope.
FIGURE: general cycling conditions of an in situ PCR, variables would depend on amplicon and enzyme systems being used.
Merits
#Supersensitive In situ detection of nucleic acids to the tune of ~1-2 copies per cell.
Sensitivity of PCR combines with the #SpatialResolution of in situ hybridization.
Has opened doors to a lot of precise applications.
Demerits
This takes an average of 48 hours to complete.
Considerably tricky to handle as it has sooo many steps and chances of going wrong in any (or all!) of them is tooo much!
Applications
This gives a visual evidence of sub-cellular localization of several genes or #pathogens.
Determination of expression of specific genes and their localization in cellular compartments.
Detection of #tumor originating tissue type and genetic alterations in tumor samples is another major application.
It has applications in #embryogenesis and #organogenesis as well.
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