A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

The overall goal of this procedure is to microdisect tissue from formal and fixed paraffin embedded slides. This is accomplished by first creating a reference image from a representative Hematin and Eoin stain slide. The second step is to place a corresponding unstained formal and fixed paraffin embedded slide onto the instrument stage and align the desired dissection area with the reference image.

Next, the dissection area is annotated and a consumable male bit is loaded with buffer and loaded into the instrument. The final step is to dissect the annotated tissue. Ultimately, a combination of RNA extraction amplification and R-T-P-C-R is used to show how the dissected tissue can be used for global gene expression analysis of mycobacterium tuberculosis within lung granulomas from infected Reeses Maca.

This method can help answer key questions in the tuberculosis research field, such as the identity of genes mycobacterium tuberculosis expresses within human-like in vivo niches during different infected states of tuberculosis. Furthermore, this data will help to explain the regulation of the pathogens, metabolic and physiologic differences within these distinct disease states. Thus generating specific drug targets.

The meso dissection instrument is equipped with the digital microscope integrated with the two ID imaging software and a joystick for controlling XY stage positions. Prior to starting this procedure, calibrate the instrument with the imaging software as described in the accompanying manuscript to create a reference image. First place a hematin and eoin stained or h and e slide on the stage and place an opaque cover on top of the slide.

Then drive the instrument by moving the joystick to the desired area of the h and d slide. Save the image generated by using the capture reference tab To align the tissue to be dissected. Click the dissect tissue option on the home screen.

In the setup tab, fill out the operator dissection accession number, reference accession number exci or barcode number dissection fluid, lot number exci size and description. Click on the find tissue tab. Import the previously saved reference image.

Place an unstained formal and fixed paraffin embedded or FFPE. Slide a five micron thickness on the stage. Move the stage to the same area as the one depicted in the reference image.

Click on the align tab. Use the mouse and associated arrows to align the reference image to the unstained image. Lastly, annotate the slide by clicking on the annotate tab.

Use the mouse to draw a circle around the desired area of the slide that will be micro dissected. Begin this procedure by filling the consumable mail bit with the desired buffer in this example PKD Buffer by pulling the plunger up and down in a fluid motion several times. Make sure to exclude air bubbles.

Raise the Z axis by pressing the Z axis button. Load the consumable mail bit into the instrument. Slide the consumable male bit into the top of the machine so that the white line on the instrument lines up with the black line on the consumable male bit.

Press the aspiration reset button to allow the plunger to be lowered into the correct position. Lower the Z axis by pressing the Z axis button again to start tissue dissection first, open the dissect tab. Next, turn the motor and aspiration speeds to one.

Check the show tracking box. This allows the user to visualize the dissected area during the dissection process to dissect. Hold the engage button as well as the aspirate button while moving the joystick in a counterclockwise direction.

Continue to dissect in a counterclockwise direction until the aspirate is full and the tab turns red. To collect the dissected sample first, press the Z axis button to raise the axis, then place a 0.5 microliter micro fuge tube under the tip of the consumable male bit, and press the aspiration control rod quickly to eject the sample into the micro fuge tube. The tissue fragment will now be contained within the micro fuge tube.

Press the aspiration control rod with increased force to eject the used consumable mal bit. The protocol demonstrated in this video was used to microdisect lung granulomas from Resus Maca infected with mycobacterium tuberculosis or MTB in various infective stages. The reference image shows an h and D stained granuloma from an actively infected reus Maca with the area of interest to be dissected outlined in green.

The middle image shows the corresponding unstained FFPE slide of five micron thickness before dissection. The areas were aligned using the software, and the correlating area of interest is outlined in green. The image on the right depicts the same unstained FFPE slide post dissection with the area dissected represented in blue.

RNA was subsequently extracted from the meso dissected granulomas at each infective state, and this table shows the results. RNA concentration was obtained using a NanoDrop. The RNA was then amplified, purified, and reverse transcribed to CD N-A-R-T-P-C-R results with respect to the 16 s ribosomal subunit confirm the presence of MTB ribosomal subunit 16 s and thus MTB.

In the microdisect samples genomic DNA from CDC 1551 strain, MTB was used as a standard Following this procedure. Other methods like D-N-A-R-N-A and protein extraction can be performed in order to answer additional questions about genomics, transcriptomics and proteomics. After watching this video, you should have a good understanding of how to microdisect FFPE samples from lung granulomas.

This procedure is not solely limited to MTB infected lung granulomas, but can be applied to lesions from other pathogens as well.