SHORT ABSTRACT
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, has recently now available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas derived from experimentally infected nonhuman primates.
LONG 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 section(1). 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 limit its efficient 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 mil bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hemotoxylin and eosin stained (H and 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 of the system 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). Tuberculosis (TB) is a major infectious disease killer of humanity worldwide and results from infection with Mtb. In a majority of individuals exposed to the aerosols of Mtb, the infection is latently limited but in at least 10 million people annually, it results in active TB disease(2). During latent infection, Mtb is contained within pathological lung lesions known as the granuloma. Hence, it has been argued that the outcome of Mtb infections is decided at the level of the granuloma(3).