2.23.0 Initial Northern Blot Analysis of msp-7 Transcription
Total RNA from ring, trophozoite, and schizont stages o f a 3D7 parasite blood
culture, separated by electrophoresis on an agarose-TBE-guanidine thiocyanate gel, was
Northern blotted onto nitrocellulose by Helen Taylor. This was probed with [a-^^P]
dATP labelled msp-7b specific to MSP-7 and analysed by autoradiography.
2.23.1 [a-^^P] dATP Labelling of msp-7b
The probe msp-7b (528 bp) (amplified and purified in Section 2.20.0) was
labelled with 30 pCi o f [a-^^P] dATP using the Prime-It II® Random Primer Labelling
Kit following the manufacturers’ instructions (Stratgene). Briefiy, 25 ng of msp-7b was
made up to 24 pi with sdw, 10 pi o f random oligonucleotides added, a small hole made
in the lid and the reaction heated at 100 °C for 5 min, then cooled to RT. To this was
added 10 pi o f 5 x *dATP primer buffer, 3 pi o f [a-^^P] dATP at 10 Ci p l'\ the contents
mixed with a pipette tip and 1 pi o f Exo(-) Klenow enzyme (5 U pl'^) added. This was
incubated at 37 °C for 5 min and the reaction stopped with 2 pi o f stop mix.
Radiolabelled probe was separated from unincorporated [a-^^P] dATP by gel
filtration through a NICK Sephadex G-50 column (Pharmacia Biotech), following the
manufacturer’s instructions. The NICK Column was equilibrated with 3 ml of
added to the column with 400 \il o f equilibration buffer. The radiolabelled probe was
eluted with an additional 400 pi o f equilibration buffer, and then denatured at 100 °C for
2 min before hybridisation to the Northern blotted RNA.
2.23.2 Analysis of RNA by Northern Hybridisation
The Northern blot was incubated at 55 °C with prehybridisation solution
consisting o f 6 x SSC, 5 x Denhardt’s reagent, 0.5 % SDS, 100 pg ml"^ denatured salmon
sperm DNA, 50 % formamide at 55 °C, with gentle rotation in a Hybaid bottle. The
denatured probe was added to the prehybridisation solution and hybridised overnight at
55 °C. Unbound probe was removed with a low stringency wash o f 2 x SSC, 0.1 % SDS,
at RT for 5 min, followed by a second higher stringency wash o f 2 x SSC, 0.1 % SDS, at
55 °C for 15 min. The blot was wrapped in Saran wrap to prevent desiccation which
would result in irreversible binding o f the probe, exposed to Kodak Biomax M Rl film at
-70 °C 0 /N and developed as before.
2.23.3 Analysis of msp-7 Transcription at Two Hour Time Points Throughout the P. falciparum 3D7 Erythrocytic Cycle
2.23.3.0 Total RNA Collected at Two Hour Time Points
Total RNA fi*om synchronised 3D7 parasites was collected at 2 hour intervals
throughout a 48 hour period. This work was done in association with Helen Taylor,
Malika Kaviratne, Irene Ling and Muni Grainger.
Mature erythrocytic schizonts o f P. falciparum strain 3D7 enriched by plasmagel
and percoll treatment were incubated with fi-esh erythrocytes for 30 min then sorbitol
treated for 30 min to lyse any remaining schizonts, giving highly synchronised rings in a
Chapter Two : Methods 97
schizonts was taken as time zero (T = 0). The highly synchronised rings were aliquoted
into 24 X 5 ml culture flasks giving a 20 % haematocrit, and cultured at 37 °C. Every 2
hours over a period o f 48 hrs, parasites in one flask were resuspended, two thin blood
smears made and Giemsa stained, and the parasites pelleted by centrifugation at 300 x g,
5 minutes, giving a 0.7 ml pellet o f infected red blood cells for each time point. The
supernatant was aspirated and the pellet resuspended in 10 pellet volumes o f TRIzol pre
warmed to 37 °C, and 1 ml aliquots o f the resuspended pellet dispensed into RNase-free
1.5 ml Eppendorf tubes, labelled with the appropriate time point, and stored at -7 0 °C
until processing.
2.23.3.1 Extraction of RNA from Collected Time Points
Total RNA was extracted from the 1 ml volumes o f each time point, where each
1 ml volume contained the equivalent o f a 100 pi pellet o f infected rbc, following the
protocol described in Section 2.2. The RNA extracted from each 1 ml sample was
resuspended in 20 pi o f formamide, the replicate samples were pooled for each time
point, prior to electrophoresis and Northern blotting.
2.23.3.2 Northern Blotting of RNA Time Points
The concentration o f RNA isolated from each time point was determined, and
equal volumes o f RNA from each time point run on an agarose-guanidine thiocyanate
gel, this was stained with ethidium bromide, photographed and then Northern blotted
(Performed by Shahid Khan). The stage o f the parasite at each time point was confirmed
by light microscopy and photography o f the giemsa stained slides.
The Northern blot was hybridised with a number o f stage specific probes
including msp-7b and stevor (Cheng et a l 1998). Twenty five ng o f purified msp-7h
amplified fi’om 3D7 gDNA, was labelled with 30 pCi o f [a-^^P] dATP using the Prime It
II Random Primer labelling kit, purified by running through a NICK column, and
denatured, essentially as described in Section 2.23.1.
The blot was prehybridised in 0.5 M sodium phosphate buffer pH 7.2, 7 % SDS
at 55 °C for 2 hours with gentle rotation in a Hybaid bottle, the denatured probe was
added to the prehybridisation buffer and hybridised overnight at 55 °C. Unbound probe
was removed with a low stringency wash in 2 x SSC, 0.1 % SDS, at RT for 15 min,
followed by a second higher stringency wash in 2 x SSC, 0.1 % SDS, at 65 °C for 15
min. The blot was covered in Saran wrap and exposed to Kodak Biomax MRl film at -70
°C and developed as before.
Hybridised probes were stripped fi"om the blot by heating the blot to 100°C in 0.1
X SSC, 0.5 % SDS, and leaving to cool at RT with gentle shaking. The stripped blot was
stored until required in Saran wrap with 2 x SSC at RT to prevent desiccation.
2.24 Western Blot Analysis of MSP-7 in Schizonts, Merozoites and Shed MSP-1