In Situ Hybridization Applicable to mRNA Species in Cultured Cells
RNA in situ hybridization (ISH) techniques allow the study of gene expression at the individual cell level in a histocytomorphological context. These techniques often involved the use of radioactive-labeled probes. However, because of resolution drawbacks limiting their applicability in the study of RNA distribution at (sub)cellular sites, these probes are replaced more frequently by hapten- or fluorophore-labeled ones. Nevertheless, radioactive ISH is still useful for detecting relatively low abundant RNA transcripts in tissue sections.
From the early 1980s, several nonisotopic-labeling methods have been developed that are most often based on the introduction of haptens (e.g., biotin and digoxigenin) or fluorochromes (e.g., fluorescein, rhodamine, coumarin, Cy3, and Cy5) that are conjugated to allyl or alkylamine-dUTP in DNA or RNA probes. Such probes provide a high spatial resolution and allow simultaneous detection of multiple RNA sequences (Dirks et al., 1991; Levsky et al., 2002) or RNA sequences together with proteins (Dirks, 1998; Snaar et al., 1999).
Direct ISH techniques, using fluorochrome-labeled probes, are used to detect relatively abundant mRNA species by conventional fluorescence microscopy. Using more advanced digital imaging microscopy, visualization of single RNA transcripts proved even possible when oligodeoxynucleotide probes were labeled with five fluorochromes per molecule and 10 oligonucleotide probes were hybridized to adjacent sequences on an RNA target (Femino et al., 1998).
Indirect ISH techniques, using haptenized probes, are often the method of choice because of their better sensitivity compared to direct fluorescence ISH approaches (Dirks et al., 1993). Furthermore, enhancement of ISH signals can be accomplished by applying multiple antibody layers or by the use of a tyramidebased detection method (Adams, 1992; Raap et al., 1995; van de Corput et al., 1998). Tyramide-based detection methods involve the use of an antihapten peroxidase-labeled antibody as a first antibody layer and a biotin, DNP, or fluorochrome tyramide that serves as a peroxidase substrate to generate and deposit many reporter molecules close to the hybridization site.
This article describes a protocol that allows sensitive detection of RNAs at cytoplasmic as well as at nuclear sites of cells that are grown or attached to microscopic slides.
II. MATERIALS AND INSTRUMENTATION
Dulbecco's modified Eagle medium without phenol red (DMEM, Cat. No. 1188-36), fetal bovine serum (FBS, Cat. No. 1050-64), l-glutamine (Cat. No. 2503-24), penicillin-streptomycin (Cat. No. 1514-22), and trypsin (Cat. No. 2509-28) are from Invitrogen Life Technologies. Salmon testes DNA (Cat. No. D-7656), dithiothreitol (DTT, Cat. No. D-0632), thimerosal (Cat. No. T-5125), polyvinylpyrrolidone (PVP, Cat. No. PVP- 40), Tween 20 (Cat. No. P-1379), mouse monoclonal antidigoxin (Cat. No. D-8156), rabbit antimouse fluorescein isothiocyanate (FITC) (Cat. No. F-7506), Streptavidin- FITC (Cat. No. S-3762), and goat antirabbit FITC (Cat. No. F-9262) are from Sigma. Ficoll PM 400 (Cat. No. 1-30-0) is from Amersham Biosciences. Bovine serum albumin fraction V (BSA, Cat. No. 44155) is from BDH. Formamide (Cat. No. 7042) and acetic acid (Cat. No. 6052) are from J. T. Baker. Amberlite MB1 ion exchanger (Cat. No. 40701) and 4,6,- diamidino-2-phenylindol 2HCl (DAPI, Cat. No. 18860) are from Serva. Acid-free formaldehyde (Cat. No. 3999) is from Merck. dATP (Cat. No. 1051 440), dCTP (Cat. No. 1051 458), dGTP (Cat. No. 1051 466), dTTP (Cat. No. 1051 482), DNase I (Cat. No. 104 158), digoxigenin-11-dUTP (Cat. No. 1093 088), sheep antidigoxigenin HRP (Cat. No. 1207 733), and blocking reagent (Cat. No. 1096 176) are from Roche. DNA polymerase I (Cat. No. M2051) is from Promega. Vectashield mounting medium is from Vector. Staining jars (100ml) for object slides (Cat. No. L4110) are from Agar Aids Ltd. TSA fluorescein system (Cat. No. NEL701), TSA tetramethylrhodamine NEL702), TSA coumarin system (Cat. No. NEL703), and TSA biotin system (Cat. No. NEL700) are from NEN.
Fluorescence ISH results were examined with an epifluorescence microscope (DM, Leica) equipped with a 100-W mercury arc lamp and a triple excitation filter for red, green, and blue excitation (Omega). Digital images were captured with a cooled CCD camera (Photometrics).
A. Labeling of DNA by Nick Translation
B. Culturing and Fixation of Cells
10x PBS (pH 7.2): Add 80g NaCl, 2g KCl, 15g Na2HPO4·2H2O, and 1.2 g KH2PO4 and adjust to 1 liter. Autoclave the solution and store at room temperature.
C. Pretreatment and Hybridization
0.1% pepsin: To make 100ml, dissolve 0.1 g pepsin in distilled water, adjust pH to 2.0, and bring to 100ml. Make this solution about 15 min before use and place in a 37°C water bath.
D. Posthybridization Washes Solutions
E. Conventional Immunocytochemical Detection
Blocking solution: To make 100ml, add 0.5 g blocking reagent and 100µl thimerosal (to prevent bacterial growth). Complete to 100ml with 1× TBS. Heat the mixture for 1h at 60°C to dissolve the blocking reagent and aliquot in 10-ml portions. Store at -20°C.
F. Tyramide Signal Amplification
10x TNT: To make 1 liter, dissolve 121.4g Tris and 87.4 g NaCl in 800ml distilled water. Ad 0.5 ml Tween 20. Mix thoroughly, adjust the pH to 7.4, and bring to a total volume of 1 liter.
The protocol just described allows sensitive detection of specific mRNAs in a variety of cell types, including cultured cells, trypanosomes (Chaves et al., 1998), malaria parasites (Vervenne et al., 1994), and blood cells. The various steps in this protocol have been optimized for detecting RNA molecules in cultured mammalian cells by trial and error (Dirks et al., 1993) and may need some adjustments for specific applications. For example, if RNA FISH is applied at the electron microscopic level of resolution, sample preparation, fixation, and pretreatment conditions
Compared to conventional immunocytochemical detection systems, the use of tyramide-based detection systems results in an at least a 10-fold increase in signal intensity (Raap et al., 1995). This is illustrated in Fig. 1, showing hybridization signals of human elongation factor (HEF) mRNA in HeLa cells and of IL-6 and G-CSF mRNA in human bladder carcinoma cells grown on microscope slides. If required, the localization properties of tyramides can be improved by the addition of dextran sulfate to the amplification diluent (Van Gijlswijk et al., 1996).
This hybridization protocol also allows bright-field microscopic visualization of hybridization signals when conjugates with peroxidase or alkaline phosphatase are used instead of fluorochrome antibody conjugates (Dirks et al., 1993).
As a positive control on the procedure, probes specific for housekeeping gene transcripts, like human elengation factor and actin mRNA, or for rRNA, such as 28S rRNA, can be used.
Finally, in order to study dynamic aspects of RNA localization, methods have been developed that allow hybridization of probes to RNAs and its subsequent visualization in living cells (Molenaar et al., 2001; Pederson, 2001).
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