Name: analysis of single-cell gene transcription by rna fluorescent in situ hybridization (fish)
Uploaded: 2012-10-07T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH at JoVE.com

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.

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.
Day 1
<ol>
 <li>Harvest 200 &mu;l of packed blood cells from a P. falciparum culture containing 2-5 % late stage IE by centrifugation ...

<ol>
	<li>Joergensen, L., Bengtsson, D. C., Bengtsson, A., Ronander, E., Berger, S. S., Turner, L., Dalgaard, M. B., Cham, G. K., Victor, M. E., Lavstsen, T., Theander, T. G., Arnot, D. E., Jensen, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+co-expression+of+two+different+PfEMP1+antigens+on+single+Plasmodium+falciparum-infected+erythrocytes+facilitates+binding+to+ICAM1+and+PECAM1.">Surface co-expression of two different PfEMP1 antigens on single Plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1.</a> PLoS Pathog. 2, 1001083 (2010).</li><li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization.">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat. Protoc. 3, 1370-1379 (2008).</li><li>Cheeseborough, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> District Laboratory Practice in Tropical Countries. , (2006).</li><li>Brolin, K. J., Ribacke, U., Nilsson, S., Ankarklev, J., Moll, K., Wahlgren, M., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+transcription+of+duplicated+var2csa+gene+copies+in+individual+Plasmodium+falciparum+parasites.">Simultaneous transcription of duplicated var2csa gene copies in individual Plasmodium falciparum parasites.</a> Genome Biol. 10, R117 (2009).</li><li>Epp, C., Li, F., Howitt, C. A., Chookajorn, T., Deitsch, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+associated+sense+and+antisense+noncoding+RNAs+are+transcribed+from+the+var+gene+family+of+virulence+genes+of+the+malaria+parasite+Plasmodium+falciparum.">Chromatin associated sense and antisense noncoding RNAs are transcribed from the var gene family of virulence genes of the malaria parasite Plasmodium falciparum.</a> RNA. 15, 116-127 (2009).</li><li>Freitas-Junior, L. H., Hernandez-Rivas, R., Ralph, S. A., Montiel-Condado, D., Ruvalcaba-Salazar, O. K., Rojas-Meza, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+heterochromatin+propagation+and+histone+acetylation+control+mutually+exclusive+expression+of+antigenic+variation+genes+in+malaria+parasites.">Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites.</a> Cell. 121, 25-36 (2005).</li><li>Obernosterer, G., Martinez, J., Alenius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locked+nucleic+acid-based+in+situ+detection+of+microRNAs+in+mouse+tissue+sections.">Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections.</a> Nature Protoc. 2, 1508-1514 (2007).</li><li>Li, F., Sonbuchner, L., Kyes, S. A., Epp, C., Deitsch, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+non-coding+RNA+are+transcribed+from+the+centromeres+of+Plasmodium+falciparum+and+are+associated+with+centromeric+chromatin.">Nuclear non-coding RNA are transcribed from the centromeres of Plasmodium falciparum and are associated with centromeric chromatin.</a> J. Biol. Chem. 283, 5692-5698 (2008).</li><li>Thompson, J., Sinden, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+Pbs21+mRNA+during+sexual+development+of+Plasmodium+berghei.">In situ detection of Pbs21 mRNA during sexual development of Plasmodium berghei.</a> Mol. Biochem. Parasitol. 68, 189-196 (1994).</li><li>Thompson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+RNA+in+blood-+and+mosquito-stage+malaria+parasites.">In situ detection of RNA in blood- and mosquito-stage malaria parasites.</a> Methods Mole Med. 72, 225-235 (2002).</li><li>Arnot, D. E., Ronander, E., Bengtsson, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progression+of+the+intra-erythrocytic+cell+cycle+of+Plasmodium+falciparum+and+the+role+of+the+centriolar+plaques+in+asynchronous+mitotic+division+during+schizogony.">The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony.</a> Int. J. Parasitol. 41, 71-80 (2011).</li><li>Osborne, C. S., Chakalova, L., Brown, K. E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J. A., Lopes, S., Reik, W., Fraser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+genes+dynamically+colocalize+to+shared+sites+of+ongoing+transcription.">Active genes dynamically colocalize to shared sites of ongoing transcription.</a> Nat. Genet. 36, 1065-1071 (2004).</li></ol>

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...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Acetic Acid </td>
 <td>Sigma/Aldrich</td>
 <td>338826-100ml</td>
 <td></td>
 </tr>
 
 <tr>
 <td>Albumax media: RPMI 1640
 Glutamine solution </td>
 <td>Lonza</td>
 <td>BE12-115F</td>
 <td>500 ml RPMI 1640
 
 5 ml glutamine solution</td>
 </tr>
 <tr>
 <td>Albumax media: Gentamycin sulphate 
 Albumax-solution</td>
 <td>Lonza</td>
 <td>BE02-012E</td>
 <td>2.5 ml gentamycin sulphate
 
 50 ml Albumax-solution</td>
 </tr>
 
 <tr>
 <td>Albumax-solution: Hypoxanthine</td>
 <td>Sigma-Aldrich</td>
 <td>H9377</td>
 <td>0.8 g Hypoxanthine </td>
 </tr>
 <tr>
 <td>Albumax-solution: AlbuMAX II</td>
 <td>Invitrogen</td>
 <td>11021-037</td>
 <td>200 g Albumax</td>
 </tr>
 <tr>
 <td>Albumax-solution:
 RPMI 1640</td>
 <td> Lonza</td>
 <td> BE12-115F</td>
 <td> 4 liter RPMI 1640
 
 
 Dissolve with magnet at max. 50 &deg;C. Filter sterilize and store at -20 &deg;C in aliquots.</td>
 </tr>
 
 <tr>
 <td>Amberlite</td>
 <td>Sigma</td>
 <td>A5710-110G</td>
 <td>Resin to deionize formamide.</td>
 </tr>
 <tr>
 <td>Anti-biotin goat pAb 
 Peroxidase 
 Conjugate</td>
 <td>Calbiochem</td>
 <td>203206</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Dig antibody</td>
 <td>Novus biologicals</td>
 <td>NB100-41330</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-fade reagent with DAPI </td>
 <td>Invitrogen</td>
 <td>P36931</td>
 <td>The mounting media has to cure for 24 hr before sealing the slide completely.</td>
 </tr>
 <tr>
 <td>Biotin RNA Labeling Mix</td>
 <td>Roche</td>
 <td>11685597910</td>
 <td></td>
 </tr>
 <tr>
 <td>Camera digital</td>
 <td>Nikon digital sight DC-F11</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Coverslips</td>
 <td>Menzel-glaser</td>
 <td>631-1570</td>
 <td></td>
 </tr>
 <tr>
 <td>culture flask 25 cm2 Nunclon surface</td>
 <td>Nunc</td>
 <td>156340</td>
 <td></td>
 </tr>
 <tr>
 <td>DEPC</td>
 <td>Fluka/Sigma</td>
 <td>32490-100ml</td>
 <td>1 ml DEPC in 1000 ml deinonized water. Add a stir bar and stir for 12 hr. Autoclave for 30 min.</td>
 </tr>
 <tr>
 <td>Digital camera</td>
 <td>Nikon digital sight DC-F11</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dynabeads Protein A</td>
 <td>Invitrogen</td>
 <td>10002D</td>
 <td></td>
 </tr>
 <tr>
 <td>DynaMaq-15 Magnet</td>
 <td>Invitrogen</td>
 <td>123-01D</td>
 <td></td>
 </tr>
 <tr>
 <td>Formamide bioultra 
 99 %</td>
 <td>Fluka/Sigma</td>
 <td>47671-1l-F</td>
 <td>Deionized formamide: 5 g of ion exchange resin per 100 ml of formamide. Stir 30 min. Filter through Whatman paper.</td>
 </tr>
 <tr>
 <td>Gelatine 0.75% solution: Gelatine</td>
 <td>Sigma-Aldrich</td>
 <td>G2500</td>
 <td>3.75 g gelatine in 500 ml RPMI 1640. Heat to 56 &deg;C to dissolve. Filter sterilize when 56 &deg;C. Store at -20 &deg;C in aliquots.</td>
 </tr>
 <tr>
 <td> Gelatine 0.75% solution: RPMI 1640</td>
 <td> Lonza</td>
 <td> BE12-115F</td>
 <td>3.75 g gelatine in 500 ml RPMI 1640. Heat to 56 &deg;C to dissolve. Filter sterilize when 56 &deg;C. Store at -20 &deg;C in aliquots.</td>
 </tr>
 <tr>
 <td>Glutamine solution: L-glutamin</td>
 <td>Sigma-Aldrich </td>
 <td>G3126</td>
 <td>14.6 g L glutamine in 500 ml 0.9 % NaCl. 
 Dissolve, filter sterilize and store at -20 &deg;C in aliquots.</td>
 </tr>
 <tr>
 <td> Glutamine solution: HCl</td>
 <td> Sigma</td>
 <td> H1758</td>
 <td>14.6 g L glutamine in 500 ml 0.9 % NaCl. 
 Dissolve, filter sterilize and store at -20 &deg;C in aliquots.</td>
 </tr>
 
 <tr>
 <td>Hybridization solution: Formamide Bio ultra 99% SSC 20x</td>
 <td>Fluka/Sigma</td>
 <td>47671-1l-F</td>
 <td>Total 20 ml, keep frozen at -20 &deg;C in aliquots 
10 ml deionized formamide</td>
 </tr>
 <tr>
 <td>Hybridization solution: Denhardt's 50x concentrate</td>
 <td>Sigma</td>
 <td>D2532</td>
 <td>5 ml 20xSSC</td>
 </tr>
 <tr>
 <td>Hybridization solution: Yeast tRNARoche blocking reagent</td>
 <td>Sigma</td>
 <td>R-6750</td>
 <td>2 ml 50x Denhardt's 
250 &mu;l 20 mg per ml yeast tRNA</td>
 </tr>
 <tr>
 <td>Hybridization solution: Salmon sperm DNA </td>
 <td>Fluka/Sigma</td>
 <td>31149-106GF</td>
 <td>0.4 g Roche blocking reagent 
1 ml of 10 mg per ml salmon sperm DNA (Critical: denature salmon spermDNA at 96 &deg;C for 5 min before adding to the hybridization solution)</td>
 </tr>
 
 <tr>
 <td>Hybridizer ThermoStar 100 HC4</td>
 <td>Quantifoil Instruments GmbH</td>
 <td>1004-0011</td>
 <td>Can be replaced by hybridization oven and RNase free hybridization chambers padded with DEPC water.</td>
 </tr>
 <tr>
 <td>Immersion oil UV transparent fluorescence free</td>
 <td>Sigma</td>
 <td>10976-1EA</td>
 <td></td>
 </tr>
 <tr>
 <td>Immunofluorescence or confocal microscope</td>
 <td>Nikon D-Eclipse TE2000C</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Nailpolish</td>
 <td>Available in any drugstore</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>PBS 20x:NaCl
 
 KCl
 
 Na2HPO4 x 2H2O
 
 KH2PO4
 DEPC deionized H2O</td>
 <td></td>
 <td></td>
 <td>Ajust pH to 7.4
 
 160 g
 
 4 g
 
 23 g
 
 4 g
 
 1 l</td>
 </tr>
 <tr>
 <td>Paraformaldehyde 4 %</td>
 <td>Fluka/chemika</td>
 <td>76240</td>
 <td>4 g PFA in 80 ml PBS/DEPC. Heat to 65 &deg;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 &deg;C.</td>
 </tr>
 <tr>
 <td>Paraformaldehyde 4 %/ Acid acetic 5 %</td>
 <td></td>
 <td></td>
 <td>950 &mu;l paraformaldhyde + 
 50 &mu;l Acetic acid</td>
 </tr>
 <tr>
 <td>Pepsin</td>
 <td>Sigma/Aldrich</td>
 <td>P7000</td>
 <td></td>
 </tr>
 <tr>
 <td>Peroxidase-conjugated anti-biotin</td>
 <td>Calbiochem</td>
 <td>203206</td>
 <td></td>
 </tr>
 
 <tr>
 <td>RBC-wash media: RPMI 1640 Glutamine solution</td>
 <td>Lonza</td>
 <td>BE12-115F</td>
 <td>500 ml RPMI 1640
 5 ml glutamine solution</td>
 </tr>
 <tr>
 <td>RBC-wash media: Gentamycin sulfate</td>
 <td> Lonza</td>
 <td> BE02-012E</td>
 <td> 2.5 ml gentamycin sulphate </td>
 </tr>
 <tr>
 <td>RNase</td>
 <td>Sigma/Aldrich</td>
 <td></td>
 <td>Make a 10 mg/ml stock solution.</td>
 </tr>
 <tr>
 <td>RNase free 1.5 ml tube</td>
 <td>Ambion</td>
 <td>AM12450</td>
 <td></td>
 </tr>
 <tr>
 <td>RNase Zap</td>
 <td>Ambion</td>
 <td>AM9780 </td>
 <td></td>
 </tr>
 <tr>
 <td>Slides 4 wells 11 mm</td>
 <td>Thermo Scientific</td>
 <td>MENZXER306W</td>
 <td></td>
 </tr>
 <tr>
 <td>SSC 20x: NaCl</td>
 <td>Sigma/Aldrich</td>
 <td>S9625</td>
 <td>3 M NaCl (175 g/l)
 0.3M Na3 citrate x H2O (88 g/l)
 Adjust to pH 7.0 with 1M HCl</td>
 </tr>
 <tr>
 <td>SSC 20x:
 Sodium citrate dehydrate</td>
 <td> Sigma/aldrich</td>
 <td> W302600</td>
 <td>3 M NaCl (175 g/l)
 0.3M Na3 citrate x H2O (88 g/l)
 Adjust to pH 7.0 with 1M HCl</td>
 </tr>
 <tr>
 <td>SSPE 20x: NaCl </td>
 <td>Sigma/Aldrich </td>
 <td>S9625</td>
 <td>175.3 g NaCl </td>
 </tr>
 <tr>
 <td>SSPE 20x: NaH2PO4</td>
 <td> Sigma/Aldrich</td>
 <td> S0751</td>
 <td> 27.6 g NaH2PO4.</td>
 </tr>
 <tr>
 <td>SSPE 20x:4 EDTA powder </td>
 <td></td>
 <td></td>
 <td>9.4 g EDTA powder 
Add DEPC water, adjust pH 7.4.
 Autoclave for 20 min</td>
 </tr>
 <tr>
 <td>TNB buffer: TNT buffer </td>
 <td></td>
 <td></td>
 <td>10 ml TNT buffer </td>
 </tr>
 <tr>
 <td>TNB buffer: Blocking reagent</td>
 <td>&nbsp; </td>
 <td></td>
 <td> 0.05 g Blocking reagent 
 To dissolve the blocking reagent, heat the solution to 60 &deg;C for one hour with stirring. Store at -20 &deg;C. (Derived from the Perkin Elmer TSA-protocol.)</td>
 </tr>
 <tr>
 <td>TNT buffer: Tris/HCl </td>
 <td>Sigma </td>
 <td>T1503</td>
 <td>1M Tris/HCl, pH 8.0</td>
 </tr>
 <tr>
 <td>TNT buffer: NaCl </td>
 <td>Sigma Aldrich</td>
 <td>S9625</td>
 <td>100 ml </td>
 </tr>
 <tr>
 <td>TNT buffer: Tween20</td>
 <td> Sigma</td>
 <td> 93773</td>
 <td> 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.)</td>
 </tr>
 <tr>
 <td>TSA plus Cyanine3/ Fluorescein system</td>
 <td>Perkin Elmer</td>
 <td>NEL753000IKT</td>
 <td>Read the protocol from the TSA Plus Fluorescence Palette System carefully before starting the experiment.</td>
 </tr>
 <tr>
 <td> Tubes 14 ml sterile </td>
 <td>Almeco - CM LAB Aps</td>
 <td>91016</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of single-cell gene transcription by rna fluorescent in situ hybridization (fish)

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.

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 &mu;m diameter) nucleus which is stained blue with DAPI. Twenty four hours after erythrocyte invasion in P. falciparum the process of schizogony, <em...

Watch this Scientific Journal Video about Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH at JoVE.com

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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