mRNA Extraction From Individual Sporocyst:
Reverse transcription of RNA and subsequent PCR amplification (RT-PCR) allows for analysing patterns of gene expression. Currently, many RNA isolation protocols are available and they are largely used. However, most of these protocols require millions of cells as starting material. In the last years, the scientific community has therefore developed protocols for RNA isolation from few cells, in particular from embryos and organisms containing around 100 cells.
One of these protocols that relies on the use of poly-T coated paramagnetic beads (Dynabeads mRNA DIRECT™ Micro kit, Dynal® Biotech), is generally used with 104 cells. We have adapted this protocol for the isolation of mRNA from individual sporocysts of Schistosoma mansoni. It is assumed that these sporocysts are composed of roughly 100 cells. Our protocol should be applicable for various organisms of similar size.
Procedure:
Parasite Culture: Schistosoma. mansoni eggs were recovered from infected Mesocrecetus auratus hamster livers 8 weeks post-infection. Livers were collected and kept in sterile saline 0.85% containing an antibiotic/ antimycotic mixture (penicillin 100 units/ml, streptomycin 0.1 mg/ml, amphotericin 0.025 µg/ml; Sigma). The livers were homogenized and the eggs were filtered and washed to obtain miracidia. Miracidia were concentrated by sedimentation on ice for 1 hour and directly submitted to in vitro transformation to obtain primary (mother) sporocysts. Miracidia were cultured in sterile chernin’s balanced salt solution (CBSS) at 26°C under atmospheric conditions.
mRNA Isolation:
The use of Dynabeads mRNA isolation is based on base pairing between the poly A residues at the 3` end of messenger RNA and the Oligo dT residues covalently coupled to the surface of the Dynabeads Oligo (dT)25.
Most steps for mRNA isolation and buffers composition were used as described in the kit. We have adapted this protocol isolate polyA mRNA from individuals sporocysts.
Living individuals sporocysts must be collected immediately into 100µl lysis binding buffer into Rnase-free tubes
Samples can be stored at -80°C for at least 4 weeks
cDNA synthesis must be performed immediately after isolation of the mRNA
cDNA Synthesis:
The following reaction mixture is prepared before beginning of mRNA isolation. For one reaction use:
11µl of sterile water in a Rnase-free tube.
1µl dNTP Mix at 10mM each (dCTP, dGTP, dATP, dTTP).
2µl of DTT as reducing agent that inhibits Rnase.
4µl 5X first-strand buffer.
1µl RnaseOUT ™ (40u/µL).
1µl (200u) of SuperScript™ II RT (Invitrogen™)
mix by flicking the tube with the fingers to create a vortex (do not vortex mechanically).
After mRNA isolation on Dynabeads, 19µl of this RT reaction mix was directly added to the tube containing the beads.
Incubate the tube for 50min at 42°C. After reverse transcription, the cDNA is fixed on the beads.
After RT, remove RT reaction mix from Dynabeads. Before PCR, the beads must be washed with 100µL of ice-cold 10mM Tris-HCl. This step removes the reverse transcriptase, that inhibits subsequent PCR amplification. Their removal improves the outcome of the PCR.
PCR Reaction:
The final reaction volume is 50µL for one PCR reaction (see Table below).
50µl of PCR mix were added directly to the tube with cDNA-RNA+beads.
The PCR program can be selected based on the composition (CG content) of the primers and the length of the expected PCR product. Nevertheless, one first denaturing step from 5min at 95°C is necessary to separate the cDNA from mRNA and help to linearise cDNA.
After PCR, 10µl of product were loaded onto a 1% agarose gel and PCR products were separated by electrophoresis (100V, 30min).
Ref:
Roger E, Grunau C, Pierce RJ, Hirai H, Gourbal B, Galinier R, Emans R, Cesari IM, Cosseau C, Mitta G: Controlled Chaos of Polymorphic Mucins in a Metazoan Parasite (Schistosoma mansoni) Interacting with Its Invertebrate Host (Biomphalaria glabrata). PLoS Negl Trop Dis 2008, 2:e330.[PubMed]