Table 3 Troubleshooting in Laser microdissection of OS regions

Histological Staining

Contamination of samples

Contamination of contact surfaces such as cryostat blade, staining solutions and tissue slides with RNase

Use gloves and follow sterile method of sample preparation. Wipe down the work surfaces and instruments with Trigene, alcohol and also possibly with RNase zap sprays. Change cryostat blade after every use. Use fresh staining solutions which are prepared in RNase free solutions for tissue staining. Use sterile PALM slides and restrict the number of tissue sections per slide to 3–4. Don’t allow the tissue sections to thaw unnecessarily as it activates the RNases in the tissue.

Unsatisfactory tissue staining resulting in difficulty in identification of tissue morphology in microscope.

Using too diluted staining solution. Incubation of tissue sections in staining solution inadequately. Thick tissue sections.

Use predetermined and optimised concentration of the staining solutions. Follow the recommended staining procedure. Increase incubation for staining.

Laser microdissection

Inadequate laser microdissection of tissue section

Thick tissue sections, tissue section placed near the margins of the slide resulting in tissue section or part of it not in the laser optical plane.

Ensure adequate dehydration of tissue section following staining procedure. This could be noted by checking for watermarks on the membrane of the PALM slide. Place tissue sections in the centre of the membrane oriented parallel with each other to prevent overlapping and folding of the sections.

Failure or misdirection of cut tissue segment to catapult in the collection tube

Tissue section not dehydrated satisfactorily.

Prior to beginning of the LMD optimise the laser settings to facilitate adequate cutting without singing or burning of the surrounding tissue seen as black frill around the cut edges.

Laser used is either out of focus or of inadequate power.

Small tissue segments are effectively catapulted in the collection tube

Large area of tissue segment dissected

Adequate dehydration of the slide could be ensured by incubating the slide in warm air incubator for approximately 5 minutes or fixing the stained slide in the ethanol bath

Tissue section not adequately dehydrated

Following LMD scan the slide surface under microscope to detect any misdirected pieces which could be redirected in collection tube by realigning the collection tube over the tissue segment and recatapultion of tissue segment with LPC laser function

Inadequate laser power

Check the collection tube cap for number of tissue segments catapulted in comparison to actual LMD segments

RNA Extraction

Inadequate RNA quality and quantity

RLT buffer leaks from the collection tube

Pooling of the RNA samples from same biological replicate

Contamination of RNA samples while processing

Variable amounts of RNA