1Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, Faculty of Health Sciences, University of Copenhagen, 2Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), 3Institute of Infection and Immunology Research, School of Biology, University of Edinburgh
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Ronander, E., Bengtsson, D. C., Joergensen, L., Jensen, A. T. R., Arnot, D. E. Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH). J. Vis. Exp. (68), e4073, doi:10.3791/4073 (2012).
Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.
1. Generation of Freshly Selected Infected Erythrocytes
For this assay, the best results are obtained when using freshly selected cultures for surface expression of PfEMP1 protein. In this particular experiment the 3D7 P. falciparum lineage was selected using specific antibodies as previously described1.
2. Preparation of Probes
The antisense RNA probes were generated from the most variable regions of the PFD1235w and the PF11_0008 var genes from P. falciparum 3D7 genomic DNA. DNA was amplified by PCR and cloned into the pSPT18 or 19 vector for transcription, respectively according to the manufacturer's description (Roche). The probes (580 base pairs (bp) and 590 bp in length) were labeled with Digoxigenin (DIG) or Biotin using a DIG RNA labeling Kit or a Biotin RNA labeling Mix, respectively. We confirmed the specificity of the probes both by Northern blot analyses and by single-label FISH analysis1.
3. Thin Smear and Fixation of Parasites Prior to In Situ Hybridization
All steps are performed in an RNase-free environment and all reagents are RNase-free or pre-treated with diethyl pyrocarbonate (DEPC) or RNase Zap (Invitrogen). The slides and cover slips should be cleaned with alcohol to remove residual manufacturing grease. It is important to perform the protocol without any pause in between steps in order to minimize the risk of nuclease contamination.
4. Hybridization of RNA Probes to Fixed Slides
5. Antibody Conjugation and Fluorescence Amplification
The following steps are based on the TSA Plus Fluorescence Palette System from Perkin Elmer. All the steps are done at room temperature.
6. Visualization of Hybridized Probes
Figure 1 illustrates a flowchart of the major steps and the time duration of the RNA FISH methodology.
A series of representative images of well and poorly preserved stained mRNA FISH experiments using single P. falciparum IE are shown in Figure 2. This intracellular protozoan has a small (1-1.5 μm diameter) nucleus which is stained blue with DAPI. Twenty four hours after erythrocyte invasion in P. falciparum the process of schizogony, i.e. nuclear division, begins. Optimal FISH detection of var gene transcription occurs at around 10-22 hr into this 48 hr intra-erythrocytic lytic cycle. Probes generally hybridize to mRNA species which appear adjacent to the main nuclear body, in what is probably nuclear envelope-associated endoplasmic reticulum. The good quality images in row A show FITC- (green) and Cyan3- (red) labeled probes corresponding to the PFD1235w and PF11_0008 var genes, respectively, in the double selected P. falciparum sub-line, 3D7 PFD1235w/PF11_0008. Co-localization of the genes is apparent but not absolute. Row B shows poor hybridization of both probes to parasites which have either degraded or largely ceased transcribing var mRNA. In row C, a good FISH hybridization is obtained but the cellular preservation is poor, as shown by badly spread and aggregating parasites.
Figure 1. RNA FISH experiment flowchart. The flowchart illustrates the main points and timing of the FISH procedure.
Figure 2. Confocal images of RNA-FISH in double selected P. falciparum indicating simultanous mRNA transcription of two different var genes labeled with either FITC (green) or Cyan3 (red) signal. DAPI staining (blue) indicates the parasite nuclear DNA. The vertical row a1, b1 and c1 show the transmission images of the parasites. Scale bar 5 μm.
RNA FISH analysis, in contrast to methods such as Northern blotting and RT-PCR, allows discrimination of specific mRNA transcripts at the single cell level. This makes it possible to discriminate between transcriptionally active and inactive cells, in this example, P. falciparum parasitic protozoa inside human red blood cells. Such whole-cell observations are often necessary and may unravel important and novel transcriptional patterns1.
Although other RNA FISH methods have been described4-10, there has been a need for development of a new and refined protocol for the study of simultaneous var mRNA transcription in P. falciparum. By using asRNA probes as detection tools, the special-temporal patterns of mRNA activity can be easily localized in the cells. In addition, asRNA probes can also be used in other experimental systems which would support their specificity1. Other critical parameters that are important when developing a new protocol includes fixation and permeabilzation of the cells. These steps were empirically defined by testing various chemical reagents as suggested by other authors5. We concluded that by combining the slow acting cross linker paraformaldehyde together with the coagulant fixative acetic acid we could obtain a good preservation of the tissue morphology. In addition, we found out that pre-blocking the cells before adding the probes was not necessary, similarly to other described RNA FISH protocols5. Furthermore, in the present protocol we use gentle washes, a mild protease (pepsin) at low amounts and a short incubation period to allow for the probe and the antibodies to diffuse into the nucleus and hybridize with the mRNA attached on the ER, minimizing the damage to surrounding membranes (Figure 2a1-2a4).
In addition, the RNA FISH and the high resolution images generated by the camera attached to the confocal microscope reveals whether a particular cell is positive for staining or not and also gives valuable information about the spatial relationships between the stained antisense RNA and the DAPI stained nucleus. As resolved in the images in Figure 2a4, the mRNA appears to be attached next to the nucleus, probably in the endoplasmic reticulum, and not on top of it, as expected in a DNA-FISH image11-12.
Studies of single cells require observations of numerous parasites and at different time points to get a statistically significant result. Thus, RNA FISH analysis is a rather time consuming technique that demands patience and considerable trial-and-error experimentation to calibrate the technique. As RNA FISH is a technique of many parameters a trouble-shooting guide to the main steps of this protocol are given in Table 1.
|Weak signal or no signal||
|Poor cellular preservation||
|The RNase treated control is positive||
|False positive detection of negative control parasites||
Table 1. Troubleshooting guidance.
No conflicts of interest declared.
The authors would like to thank Michael Alifrangis and Ulla Abildtrup for genotyping of parasites and Christina Holm for excellent technical assistance. This work was funded by Howard Hughes Medical Institute (grant 55005511), The Lundbeck Foundation (grant R9-A840) and by the Niels Bohr Foundation.
|Albumax media: RPMI 1640 Glutamine solution||Lonza||BE12-115F||500 ml RPMI 1640
5 ml glutamine solution
|Albumax media: Gentamycin sulphate Albumax-solution||Lonza||BE02-012E||2.5 ml gentamycin sulphate
50 ml Albumax-solution
|Albumax-solution: Hypoxanthine||Sigma-Aldrich||H9377||0.8 g Hypoxanthine|
|Albumax-solution: AlbuMAX II||Invitrogen||11021-037||200 g Albumax|
|Albumax-solution: RPMI 1640||Lonza||BE12-115F|| 4 liter RPMI 1640
Dissolve with magnet at max. 50 °C. Filter sterilize and store at -20 °C in aliquots.
|Amberlite||Sigma||A5710-110G||Resin to deionize formamide.|
|Anti-biotin goat pAb Peroxidase Conjugate||Calbiochem||203206|
|Anti-Dig antibody||Novus biologicals||NB100-41330|
|Anti-fade reagent with DAPI||Invitrogen||P36931||The mounting media has to cure for 24 hr before sealing the slide completely.|
|Biotin RNA Labeling Mix||Roche||11685597910|
|Camera digital||Nikon digital sight DC-F11|
|culture flask 25 cm2 Nunclon surface||Nunc||156340|
|DEPC||Fluka/Sigma||32490-100ml||1 ml DEPC in 1000 ml deinonized water. Add a stir bar and stir for 12 hr. Autoclave for 30 min.|
|Digital camera||Nikon digital sight DC-F11|
|Dynabeads Protein A||Invitrogen||10002D|
|Formamide bioultra 99 %||Fluka/Sigma||47671-1l-F||Deionized formamide: 5 g of ion exchange resin per 100 ml of formamide. Stir 30 min. Filter through Whatman paper.|
|Gelatine 0.75% solution: Gelatine||Sigma-Aldrich||G2500||3.75 g gelatine in 500 ml RPMI 1640. Heat to 56 °C to dissolve. Filter sterilize when 56 °C. Store at -20 °C in aliquots.|
|Gelatine 0.75% solution: RPMI 1640||Lonza||BE12-115F||3.75 g gelatine in 500 ml RPMI 1640. Heat to 56 °C to dissolve. Filter sterilize when 56 °C. Store at -20 °C in aliquots.|
|Glutamine solution: L-glutamin||Sigma-Aldrich||G3126||14.6 g L glutamine in 500 ml 0.9 % NaCl. Dissolve, filter sterilize and store at -20 °C in aliquots.|
|Glutamine solution: HCl||Sigma||H1758||14.6 g L glutamine in 500 ml 0.9 % NaCl. Dissolve, filter sterilize and store at -20 °C in aliquots.|
|Hybridization solution: Formamide Bio ultra 99% SSC 20x||Fluka/Sigma||47671-1l-F||Total 20 ml, keep frozen at -20 °C in aliquots
10 ml deionized formamide
|Hybridization solution: Denhardt's 50x concentrate||Sigma||D2532||5 ml 20xSSC|
|Hybridization solution: Yeast tRNARoche blocking reagent||Sigma||R-6750||2 ml 50x Denhardt's
250 μl 20 mg per ml yeast tRNA
|Hybridization solution: Salmon sperm DNA||Fluka/Sigma||31149-106GF||0.4 g Roche blocking reagent
1 ml of 10 mg per ml salmon sperm DNA (Critical: denature salmon spermDNA at 96 °C for 5 min before adding to the hybridization solution)
|Hybridizer ThermoStar 100 HC4||Quantifoil Instruments GmbH||1004-0011||Can be replaced by hybridization oven and RNase free hybridization chambers padded with DEPC water.|
|Immersion oil UV transparent fluorescence free||Sigma||10976-1EA|
|Immunofluorescence or confocal microscope||Nikon D-Eclipse TE2000C|
|Nailpolish||Available in any drugstore|
Na2HPO4 x 2H2O
KH2PO4 DEPC deionized H2O
|Ajust pH to 7.4
|Paraformaldehyde 4 %||Fluka/chemika||76240||4 g PFA in 80 ml PBS/DEPC. Heat to 65 °C until the PFA dissolves. Add 20 ml PBS, allow the solution to cool. Adjust the pH to 7.4. Filter. Store in aliquots at -20 °C.|
|Paraformaldehyde 4 %/ Acid acetic 5 %||950 μl paraformaldhyde + 50 μl Acetic acid|
|RBC-wash media: RPMI 1640 Glutamine solution||Lonza||BE12-115F||500 ml RPMI 1640 5 ml glutamine solution|
|RBC-wash media: Gentamycin sulfate||Lonza||BE02-012E||2.5 ml gentamycin sulphate|
|RNase||Sigma/Aldrich||Make a 10 mg/ml stock solution.|
|RNase free 1.5 ml tube||Ambion||AM12450|
|Slides 4 wells 11 mm||Thermo Scientific||MENZXER306W|
|SSC 20x: NaCl||Sigma/Aldrich||S9625||3 M NaCl (175 g/l) 0.3M Na3 citrate x H2O (88 g/l) Adjust to pH 7.0 with 1M HCl|
|SSC 20x: Sodium citrate dehydrate||Sigma/aldrich||W302600||3 M NaCl (175 g/l) 0.3M Na3 citrate x H2O (88 g/l) Adjust to pH 7.0 with 1M HCl|
|SSPE 20x: NaCl||Sigma/Aldrich||S9625||175.3 g NaCl|
|SSPE 20x: NaH2PO4||Sigma/Aldrich||S0751||27.6 g NaH2PO4.|
|SSPE 20x:4 EDTA powder||9.4 g EDTA powder
Add DEPC water, adjust pH 7.4. Autoclave for 20 min
|TNB buffer: TNT buffer||10 ml TNT buffer|
|TNB buffer: Blocking reagent|| 0.05 g Blocking reagent
To dissolve the blocking reagent, heat the solution to 60 °C for one hour with stirring. Store at -20 °C. (Derived from the Perkin Elmer TSA-protocol.)
|TNT buffer: Tris/HCl||Sigma||T1503||1M Tris/HCl, pH 8.0|
|TNT buffer: NaCl||Sigma Aldrich||S9625||100 ml
|TNT buffer: Tween20||Sigma||93773|| 5 M NaCl 30 ml
1 ml DEPC dH2O 869 Ml
Adjust pH to 7.5 at room temperature. (Derived from the Perkin Elmer TSA-protocol.)
|TSA plus Cyanine3/ Fluorescein system||Perkin Elmer||NEL753000IKT||Read the protocol from the TSA Plus Fluorescence Palette System carefully before starting the experiment.|
|Tubes 14 ml sterile||Almeco - CM LAB Aps||91016|