Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies,
DNA assessment or chromosomal analysis. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of
RNA, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure
RNA preservation. Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies. Small pieces of pancreas of about 0.5-1 cm(3) were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled
2-Methylbutane, and stored at -80 °C until sectioning. Forty serial sections of 10 μm thickness were cut on a cryostat under a -20 °C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 °C. Immediately before the
laser microdissection procedure, sections were fixed in
ice cold, freshly prepared 70%
ethanol for 30 sec, washed by 5-6
dips in
ice cold DEPC-treated water, and dehydrated by two one-minute incubations in
ice cold 100%
ethanol followed by
xylene (which is used for tissue
dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in
xylene, were performed using
ice-cold
reagents - a modification over a previously described protocol. utilization of
ice cold
reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto
Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 μl lysis
buffer. Each single
RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by
RNA extraction using the Pico Pure
RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with
type 2 diabetes.