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....

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 aspir....

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