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

Summary

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Abstract

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section¹. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

Introduction

Samples have traditionally been manually microdissected from either whole tissue or slides using a needle and scalpel. This necessitates a clear separation between the tissue section of interest and surrounding tissues sections². With the current advancements in molecular profiling technology, there has been an increasing need to assess tissue at the cellular level. Due to the limitations of manual microdissection, techniques including LCM were established to allow greater isolation precision. This technique allows the researcher to isolate specific cell populations from various cell and slide types, which can then be used for downstream assays such as gene expression profiling. Although very effective for downstream assays, LCM is not without limitations. First, LCM is an expensive and time consuming process. Additionally, due to the unstable nature of RNA, it is often challenging to obtain high quality RNA from LCM samples². Due to the disadvantages of LCM, new advances in microdissection technology are still needed to make it more accessible to a greater number of researchers in an affordable and time sensitive manner.

One such advancement in microdissection technology now available is a technique known as mesodissection. In this technique a machine is used to mill the annotated tissue section of interest and aspirates it into a consumable mill bit³. Next, this sample can be aspirated into a collection tube and used for downstream applications. The advantages of this system are that it is significantly less expensive and time sensitive. In our experience, the system allows the dissection of a lung granuloma tissue section in ten minutes. On the other hand, traditional LCM would likely require hours to complete the process. The system allows the operator to load a reference slide to use as a comparison as well as tool for annotation. Additionally a report is generated outlining the area of the slide that was dissected. There are two main disadvantages to mesodissection. Although effective at extracting multiple cells, it is challenging to isolate a single cell. In addition, the precision of the image generated is not as clear as if using other imaging microscopes.

Tuberculosis (TB) is a major infectious disease killer of humanity worldwide and results from infection with Mtb. In a majority of individuals exposed to aerosols of Mtb, the infection is latently limited. In at least 10 million people annually, it results in active TB disease⁴. During latent infection, Mtb is contained within pathological lung lesions known as granulomas. Hence it has been argued that the outcome of Mtb infection is decided at the level of the granuloma⁵.

Here we demonstrate how mesodissection can be used to microdissect granulomas caused by Mtb. The slides used are from FFPE lung tissue from infected rhesus macaques. For the purpose of this demonstration, we will dissect granulomas in their entirety. We also demonstrate that RNA can be extracted from the recovered tissue. This technique can be applied to tissue samples from various other specimens and then used for a variety of downstream assays.

Protocol

1. Calibrate Mesodissection Instrument with 2iD Imaging Software

The 2iD imaging software will be referred to as either the software or the program for the remainder of this article. This step is needed to correctly track the image as well as align multiple slide frames.

1. Turn on instrument and computer.
2. Open software and select “calibrate instrument”.
3. Calibrate stage travel by using the joystick. Move stage to upper left position. Press “set” under step 1 in program.
4. Move stage to lower right position. Press “set” under step 2 in program.
5. Calibrate aspiration by first pressing “Z-button” to raise head assembly. Press “aspirate pulse” button and “dissection” button on the joystick until plunger control rod reaches top position. Press “set” under step 3 of program.
6. Press “aspirate reset” button. Once the plunger stops at the bottom position press “set” in step 4.
7. Click on the “scale tab”.
8. Place calibration ruler on blank side.
9. Focus microscope if needed.
10. Draw line between the two scale bars using mouse.
11. Enter distance in box labeled step 2. Press “compute” button.
12. Click on “crosshair” tab. Turn motor speed to 1.
13. Insert xScisor into head assembly. Lower head assembly into z-axis ready position by pressing “z-axis” button. NOTE: The head assembly is the large structure on the top the dissection instrument. The xScisor will be referred to as a consumable mill bit for the remainder of this article.
14. Click “dissection engage” button on joystick. NOTE: This button is located on the top of the joystick.
15. Define center of rotation visually. To do this, move center of crosshair over center of rotation using defined parameter in step 1 of program. Use the up and down arrows in the “x” and “y-axis” bars to adjust the pixels to align crosshair. Click the “done” tab.
16. Click on “home” icon. NOTE: This is represented by a house image located in the upper right hand corner.

2. Create Reference Image

1. Place H&E slide on stage. Place opaque cover on top of slide. Drive the instrument by moving the joystick to the desired area of the H&E slide. Save the image generated. NOTE: Either the “capture reference” tab or “dissect” tab can be used to complete this step.

3. Align Tissue for Dissection

1. Click the “dissect tissue” option on the home screen.
2. Fill out the “operator,” “dissection accession number,” “reference accession number,” “xScisor barcode number,” “dissection fluid lot number,” “xScisor size” and “description” in the “setup” tab.
3. Click on the “find tissue” tab.
4. Import previously saved reference image.
5. Place an unstained FFPE slide of 5 micron thickness on the stage. Move stage to the same area as the one depicted in the reference image. Click on “insert image” tab.
6. Click on “align” tab. Use the mouse and associated arrows to align the reference image to the unstained image.

4. Annotate Slide

1. Click on the “annotate” tab.
2. Use the mouse to draw a circle around the desired area of the slide that will be microdissected.

5. Load Consumable Mill Bit

1. Fill consumable mill bit with desired buffer, here PKD buffer, by pulling the plunger up and down in a fluid motion several times. Make sure to exclude air bubbles.
2. Raise Z-axis by pressing “Z-axis button”.
3. Load consumable mill bit into machine. Do this by sliding the consumable mill bit into the top of the machine so that the white line on the instrument lines up with the black line on the consumable mill bit. Press “aspiration reset” button to allow plunger to be lowered into the correct position. NOTE: It should be easy to slide the consumable mill bit. If it is not, make sure the notches are correctly aligned.
4. Lower Z-axis by again pressing “Z-axis” button.

6. Dissect Tissue (Figure 4)

1. Open “dissect” tab.
2. Turn motor and aspiration speeds to 1.
3. Check “show tracking” box. Note: This allows the user to visualize the dissected area during the dissection process.
4. Once ready to dissect, continue to hold “engage” button as well as “aspirate” button while moving joystick in a counterclockwise direction to dissect tissue.
5. Continue to dissect in a counterclockwise direction until the aspirate is “full” tab turns red.

7. Empty and Remove Consumable Mill Bit

1. Place a 0.5 µl microfuge tube under the tip of the consumable mill bit.
2. Press “aspiration” control rod quickly to eject sample into microfuge tube.
3. Press with increased force to eject used consumable mill bit.
4. Rinse tissue fragment by pulling plunger back. Push plunger forward to express remaining sample into consumable mill bit. The tissue fragment will now be contained within the microfuge tube.

8. Lyse Sample with Proteinase K Digestion Followed by Heat Treatment

1. Add 70 µl TE pH 8.5 containing 5 µg of proteinase K to recovered tissue sample.
2. Place sample into programmable heater-shaker.
3. Complete digestion using programmable heater-shaker at the following setting: 60 ˚C for 30 min at 1,500 rpm; 82 ˚C for 15 min at 1,500 rpm; and 25 ˚C for 1 min at 450 rpm. The sample is now ready for downstream applications.

Results

The aforementioned protocol shows how to use a new mesodissection technique to extract RNA from FFPE tissue slides. This protocol’s efficacy is shown through FFPE slides of lung granulomas from NHP infected with Mtb in various infective stages. Figures 1-3 are images of the instrument. Figure 4 depicts how the dissection process occurs and the resulting image generated by the software. Table 1 shows the results of RNA extraction with a granuloma from a NHP at ea...

Discussion

Mesodissection is a technique that can be applied to RNA extraction from pathologic lesion slides caused by a vast array of pathogens. It is a necessity that the user tracks the image and aligns the image correctly. To accomplish this, the instrument must be calibrated following the directions on the imaging software. When dissecting, if the slide and area of interest being dissected do not align, the user must recalibrate the instrument. Another critical step in the dissection process is to load the consumable mill bit ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MeSectr	AvanSci Bio		Mesodissector
400 nm xScisors	AvanSci Bio		Other sizes available
THOR	AvanSci Bio		Programmable Heater-Shaker
NanoDrop2000	ThermoScientific	ND-2000
RNeasy FFPE extraction kit	Qiagen	73504
Ovation RNA-Seq FFPE System	Nugen	7150
QIAquick PCR Purification Kit	Qiagen	28104