The overall goal of the following procedure is to increase the sensitivity for detecting cytomegalo viral DNA in gastrointestinal biopsies, particularly biopsies that test equivocal with conventional staining methods. This is achieved by first cutting and collecting scrolls of formal and fixed paraffin embedded gastrointestinal biopsy tissue. Next DNA is extracted from the tissue scrolls for amplification.
The specific primers used selectively amplify any cytomegalo viral DNA. When conventional methods used on gastrointestinal biopsy tissue demonstrate equivocal results, this sensitive method can detect cytomegalo viral DNA. The main advantage of this technique over other methods in the field, such as hematin, andin, and immunohistochemistry, is that this method does not involve the interpretation of non-class appearing staining patterns On the slide demonstrating cutting of gastrointestinal biopsy tissue will be Zainab van Horn, a histo technologist in the laboratory demonstrating the nucleic acid extraction, as well as the R-T-P-C-R setup in the laboratory will be.
Rebecca Jocelyn For this protocol. Have ready FFPE GI biopsy tissue blocks sitting on crushed ice. First label a micro fuge tube for each sample block with the tissue block's identification number at the microtome.
Ensure the hand wheel is locked and see the tissue block into the cassette clamp. Make sure you are using a new blade In order to prevent cross-contamination between specimens. It's crucial that you use an unused blade for your first specimen.
Switch to another blade in between specimens and to decontaminate the microtome in between specimens. Next, align the block to the knife angle and adjust the section thickness to 10 microns. Now unlock the hand wheel and advance the block toward the blade.
Cut five sections of the block at 10 micron intervals allowing each section to roll into a scroll of tissue. Ensure that each scroll contains an adequate representation of the desired biopsy tissue. When the five scrolls are cut, lock the hand wheel.
When transferring the five scrolls to the labeled centrifuge tube, ensure the forceps touch only the paraffin and not the tissue promptly. Cap the tube, then remove the block from the cassette clamp to collect scrolls from the next sample. First, remove and discard the blade.
And before loading a new unused blade decontaminate any surfaces that may have contacted the previous sample. Until the samples are further processed, they can be stored at room temperature. The following protocol is adapted from the FFPE tissue DNA extraction kit.
Be sure to consult the material safety data sheet before proceeding. Begin by adding a milliliter of xylene into each sample tube and then vortexing them vigorously for 10 seconds. Next, follow the manufacturer's instructions with the following modifications.
First, add six microliters of the internal control to each tube. Second, following the A TL proteinase K lysis incubation at 56 degrees Celsius for one hour. Incubate the samples at 99 degrees Celsius for 30 minutes rather than at 90 degrees Celsius for one hour.
Third, when completing the elucian step, instead of buffer a TE carefully apply 50 microliters of distilled DNAs and RNAs free water at the center of the membrane of the Elucian column. And lastly, all the centrifugation steps should be performed at room temperature. Set up the reactions in a clean hood that has no amplification product contaminants from the kit.
Remove enough master vials and magnesium solution for the samples plus five controls. Each master vial contains enough reagent for 12 tests. After thawing, vortex the master vials and briefly spin them down at maximum speed.
Next, following the PCR worksheet, combine the magnesium solution and master mix. Gently mix the solutions by pipetting placing them in the cooling block. Add 15 microliters of the mix to the test capillaries in the cooling block, except at position five.
This capillary is set aside for the quantitation standard. Move the cooling block containing the capillaries and master mix with magnesium from the clean hood to a separate processing hood. Here add two microliters of internal control to the remaining 30 microliters of master mix.
Mix the solution by pipetting up and down and eloquent 15 microliters of this mixture into the capillary at position five. Then add 10 microliters of water control DNA or sample DNA into the appropriate. Once loaded, cap the capillaries with the capping tool at the capillary centrifuge.
Transfer the capillaries from the cooling block to the corresponding position in the carousel rack. Then load the carousel into the centrifuge and run the cycle after the spin. Transfer the carousel to the real-time PCR machine and run the reaction later during analysis.
View the data using absolute quantification to ensure that a curve is available for making quantifications a total of 228. Tissue blocks were tested by QPCR 91. Blocks were CMV positive cases based on histology and positive immunohistochemistry.
18 blocks had equivocal C-M-V-I-H-C. The other 79 were negative controls. CMV positive cases demonstrated.
Typical CMV viral inclusions and positive IHC staining, or at least one of the two. The equivocal cases demonstrated rare non-class staining patterns, whereas negative controls showed no suspicious histology or IHC staining of the 91 tissues positive for CMV 88. Tested positive by QPCR of the 71 negative controls one tested positive for CMV by QPCR.
A.Discussion on the biopsies of the equivocal C-M-V-I-H-C staining is provided in the text protocol. After watching this video, you should have a good understanding of how to perform DNA extraction from formal and fixed paraffin embedded GI biopsy tissue, and then the subsequent detection of cytomegalo viral DNA by real-time PCR.
