RNA Quality after Different Tissue Sample Preparation

To compare untreated tissue samples with fixed and stained tissue samples, 3 cryo-preserved murine brains of known RNA integrity (RIN) were used. After cryo-preservation at –80° C, these were transferred to a cryo-microtome and incubated at –35 °C for 45 min. Then the tissue was equilibrated at –18° C for 30 min and cut into 12 µm sections at –18° C by cryo-sectioning. The murine-brain sections were divided into two groups which were treated in the following ways. One, the control group, was directly frozen at –80° C. The other group was applied to an RNase-free, UV-treated PEN slide. This was done by briefly thawing the sections at room temperature and fixing them for 2 min in 75% ethanol cooled to –20° C. After fixation, they were immediately stained with drops of sterile-filtered cresyl violet (CV) dye solution (5% cresyl violet in pure ethanol) and incubated for 45 secs with a slight to-and-fro movement. After this staining procedure, the dye solution was briefly drained off and the slide was immersed in a sequence of ethanol solutions with increasing concentrations (75%, 95%, and 100%) for 4 secs each and then finally fixed in 100% ethanol (test group). Following this fixing and staining procedure, the slide was placed in a drying chamber with silica gel for 45 mins at room temperature and then stored at –80° C in a dry box pending laser microdissection.

After fixing and staining, the applied tissue was isolated using the gentle and contamination-free technique of UV laser microdissection  (UV LMD) [2]. This was done by marking the tissue and dividing it into rectangles using the 10x objective of the microscope before completely ablating it with the “Draw + Cut” mode using a laser beam of sufficient energy. The dissectates were collected in 0.5 ml RNase-free caps to allow purification of the RNA content. These were stored at –80° C pending RNA isolation.

Both groups – the untreated samples stored at –80° C and the fixed, stained and laser-microdissected samples – were thawed in parallel and treated with the same reagents simultaneously. The RNeasy Mini Kit® of Qiagen was used for purification, following the manufacturer’s instructions. The purified RNA was eluted with 30-µl RNase-free water into a fresh RNase-free test tube.

To compare the RNA quality of native cryo-tissue sections with that of CV-stained, EtOH-fixed, laser-microdissected tissue sections, RNA from each of the differently treated samples was applied to the same RNA nanochip (Agilent Bioanalyzer). The RNA nanochip separates RNA samples by capillary gel electrophoresis and grades the quality of the samples according to the way they move on the chip. As mentioned above, the quality is expressed as a RIN value where 10 indicates completely intact RNA and 1 completely degraded RNA.
The RIN analysis conducted here (sample measurement in Fig. 2) showed that the RIN values of RNA from directly frozen native tissue were not much different from those treated according to the UV-LMD standard protocol.

Altogether, it was shown that UV laser microdissection with the method described [2] does not significantly impair the RNA quality of cryo-preserved brain tissue. Such a technique offers an appropriate method of sample preparation and non-destructive isolation of single cells to obtain RNA of consistent quality.