Analysis of var expression in cultured parasites
RNA isolation
1. Start with a 20 ml culture of synchronized parasites at late-ring stage.
2. Spin down cells, (4000 rpm, 4 min) re-suspend in 10 mls PBS, lyse RBCs by adding
100 μl 10% saponin.
3. Spin down parasite pellet, wash once with PBS, re-suspend in 10 ml PBS.
4. Spin down, aspirate supernatant, and take up parasite pellet in 0.75 ml room
temperature Trizol in 1.5 ml eppendorf tubes.
5. Add 0.15 ml chloroform, vortex and leave at room temp for 5 min. Microcentrifuge
for 15 minutes at 4o C, max speed.
6. Recover aqueous phase and “clean” RNA using Invitrogen Pure link micro to midi
RNA kit. Briefly:
A. Dilute aqueous phase 1:1:1 with lysis buffer (+β merc.) and 100% EtOH.
B. Put on purification column and spin.
C. Wash twice with wash buffer II followed by extra spin.
D. Elute RNA in 30 μl water.
7. Take RNA concentration.
DNAse Treatment
1. Assemble the following reaction:
A. 800 ng total RNA
B. 2 μl 10X DNAse I reaction buffer
C. 2 μl DNAse I, Amp grade, 1 U/μl
D. DEPC water to final volume of 20 μl
2. Incubate at room temp for 30 minutes.
3. Inactivate DNAse I by addition of 2μl of 25 mM EDTA, incubate at 65o C for 10 min.
4. Sample ready for cDNA synthesis.
cDNA synthesis (Invitrogen superscript system)
1. Thaw 5X first strand buffer immediately before reaction and re-freeze immediately.
2. Add the following components directly to DNase treated RNA:
A. 2 μl or 50-250 ng random primers
B. 2 μl 10 mM dNTP
C. sterile water to a final volume of 26 μl
3. Heat to 65o for 5 min., chill on ice.
4. Spin down quickly and add:
A. 8 μl 5X first-strand buffer
B. 4 μl 0.1 M DTT
C. 2 μl RNAse out
5. Mix gently and incubate at 42o C for 2 min.
6. Add 2 μl (200 units) of Superscript II RT, mix by pipetting gently.
7. Incubate at room temp for 10 min.
8. Incubate at 42o C for 50 min.
9. Inactivate reaction by heating to 70o for 15 min.
Analysis of expression of entire var gene family
For 3D7, the primer set of Salanti et al. (Mol. Micro., vol 49, pg 179-191) is used to assay
for expression of the entire var gene family. Slight modifications to this primer set are
described by Dzikowski et al. (PLoS Pathogens, vol 2, E22). A primer set is also
available for the all of the var genes in the IT parasite line from Joe Smith (SBRI).
1. A 25 mM stock solution (100X) is made for each primer.
2. A working stock (10X) of primers is made for each var gene to be assayed (59 var
genes in 3D7 plus several control genes). This is done by adding 10 μl of both the
forward and reverse primers to 80 μl water.
3. Each individual reaction consists of:
A. 10 μl Cybergreen
B. 4 μl primers
C. 2 μl cDNA
D. 4 μl water
To assay the entire var family, a master mix is made that includes the above
ingredients minus the primers in a large enough volume to accommodate all 59 var genes
plus controls. 16 μl aliquots are then distributed into the wells of a real-time PCR plate,
and individual primer pairs added to each well.
PCR conditions
1. 50o 2 min.
2. 95o 3 min.
3. 95o 15 sec.
4. 54o 40 sec.
5. 60o 1 min.
6. repeat 3-5 40 cycles.
7. optional: add dissociation step.