Table 2.

Troubleshooting.

Problems	Possible reason	Solution
LCM (Step 7)
No cells captured in LCM cap.	Tissue section mounted on a charged (plus) slide.	Mount the tissue section on uncharged slides for LCM.
	Tissue section on slide not sufficiently dry.	Change solutions including ethanol and xylenes. Now increase the times of dehydration in ethanol 95%, 100% and xylene at 2 minutes each bath.
Contamination of LCM cap with non-dissected extra tissue.	Tissue section could be too dry.	Check the dissected cells in the cap under the microscope. If there is extra tissue, remove it with a sterile adhesive tape or similar note paper. Gently place the cap with the film on the sticky border of the sterile adhesive tape or similar note paper a couple of times. Eliminate the used sterile adhesive tape or similar note paper. Now, check the cap again under the microscope. The extra tissue will be removed from the cap (attached to the sticky paper) while the dissected cells will remain embedded in the film and will be not removed by the sticky paper.
RNA Quantity and Quality (Steps 49 and 53)
No quantity or quality data reported by NanoDrop or Bioanalyzer analysis, respectively.	Tissue section stored in freezer too long.	Use tissue section within 2 weeks of sectioning and freezing.
	RNase rich tissue (e.g. pancreas, duodenum, colon, etc.).	Stain and dissect as quickly as possible. Add RNase inhibitors to staining solutions.
	Cell not captured by LCM in the cap.	Always check lifting of captured cells on the cap under the microscope after LCM before proceeding to RNA extraction.
	Nanogram quantity of sample appropriate for the Bioanalyzer chip chosen (Pico v. Nano)	Use the appropriate chip for the quantity of total RNA determined by NanoDrop.
	Washed RNA through the column during RNA extraction	Pool all flow-through and re-extract all flow through and recover the RNA.
	Total RNA not aliquotted appropriately leading to multiple free-thaw degradation.	Aliquot total RNA prior to initial storage at −80°C
	Tissue block assessment by scraping tissue section and performing RNA extraction, quantity, and quality assessments prior to microdissection was not done.	Perform tissue block assessment prior to microdissection.
Low RIN number.	Tissue or RNA degradation	Continue with qRT-PCR analysis. Use random hexamers in RT step to minimize effects of degradation. Determine acceptable RIN range for the tissue type being used in the study. For prostate, esophagus and urethra, we have found that even low RINs (RIN >2) produce specific repeatable qRT- PCR results.
qRT-PCR (Step 67)
No CT values reported.	Poor quality and quantity or total RNA template used for RT.	See above RNA Quantity and Quality section.
	RT failed to generate cDNA.	Check positive control. If positive control for a particular primer/probe set did generate CT values and the endogenous control gene analyzed for the sample generated CT values, then it can be concluded that cDNA was generated and the sample may not be expressing the transcript of interest within qRT-PCR detectable quantities. If not, repeat RT.
	Only ran qPCR for 40 cycles.	Use RT generated cDNA to run qPCR for 50 cycles.
No CT values reported in one paired sample and not the other.	Identical number of LCM shots/number of cells not collected for RNA extraction.	Use comparable number of cells/laser shots for RNA extraction and downstream qRT-PCR. If comparable numbers of cells and total RNA were used for downstream qRT-PCR, transcript in one sample (e.g. diseased sample or normal sample) may not be expressed within detectable levels. At this point it is imperative to ascertain that the endogenous control qRT-PCR for that sample did generate a CT value. If not, see No CT values reported section above.
Analysis (Step 68)
Under expression or down regulation of gene expression unable to be determined.	ΔΔCT value is positive.	Multiply positive ΔΔCT value by 1/x. Then perform 2−ΔΔCT fold change calculation using this number. This fold change value will represent the under expression compared to the calibrator.