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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Research

JoVE Journal

Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...