Problem	Possible cause	Recommendation
Weak signal or no signal	Low mRNA expression. mRNA degradation. Probe is degraded, in-efficiently labeled or too low in concentration. Too high hybridization temperature. Too long pepsin treatment. Cells are not permeabilized enough.	Check the protein expression by FACS. Work under RNAse free conditions and use only freshly prepared solutions. Do a quality check of the labeled probe on gel and compare to a labeled control RNA. Make a titration series of the probe concentration in FISH. Reduce the hybridization temperature stepwise. Reduce the pepsin treatment length. Prolong the pepsin treatment length or try another detergent.
Poor cellular preservation	Too high parasitaemia or stressed parasites. Too thick films. Badly cleaned slides. The cells on the slides are deattached.	Use a healthy parasite culture by keeping the parasitemia under 10 % and changing the cell media every day. Dilute the parasite culture. Clean the slides with alcohol and wait at least 10 min for the slides to dry. Use freshly prepared para-formaldehyde and/or increase the paraformaldehyde incubation length.
High Background	Too high probe concentration. Insufficient washing of parasites. Bursting shizonts. Insufficient post hybridization washings. Blocking buffer is contaminated or inefficient.	Make a titration series of the probe concentration in FISH. Wash the parasites twice before preparing the thin films. Synchronize the culture more tightly. Increase the number and/or length of washes. Prepare new blocking buffer. Try other blocking reagent, concentration or prolong the blocking period
The RNase treated control is positive	The Rnase solution is inactive. Too low RNase concentration. The incubation was too short.	Prepare a new Rnase solution. Increase the concentration. Make a titration series. Prolong the RNAse treatment period.
False positive detection of negative control parasites	The probe is not specific. Parasites do not express the mRNA detected by the probe.	Check the specificity of the probes. Check that the parasite line used is the right one and freshly selected.