细胞中RNA 荧光原位杂交.docx

ProtocolRNA FISH on Cultured Cells in Interphase1. Ng Karen1 and 2. Anton Wutz+ Author Affiliations1. Research Institute of Molecular Pathology (IMP), 1030 Vienna, Austria1. 1Corresponding author (ngimp.univie.ac.at) Next SectionINTRODUCTIONFluorescence in situ hybridization (FISH) has become a widely used method in genome and molecular genetic studies. The technique is highly versatile and has been adapted to carry out genome-wide screenings, microarray quantifications, cancer cytogenetics analysis, and RNA expression and localization studies. The study of intracellular RNA localization using RNA FISH provides insights into the in situ physical characteristics of transcription and intracellular RNA transport in individual cells. In our lab, we use RNA FISH to detect the localization of Xist RNA, a nuclear noncoding transcript that coats the entire chromosome from which it is transcribed. The advantage of using RNA FISH in our case is to extract precise molecular information directly in the context of cellular structure. The RNA FISH technique requires the generation of a labeled probe, hybridization of the probe to a fixed sample, and detection of the labeled probe using microscopy. Previous SectionNext SectionRELATED INFORMATIONFigure 1 illustrates the use of the RNA FISH protocol to detect Xist in transgenic XXz mouse ES cells. Bray-Ward (2002) provides additional information on the FISH technique. View larger version: In this page In a new windowFigure 1. Xist RNA FISH in transgenic Xz mouse ES cells. The in situ hybridization is performed as described in the protocol. Xz cells express Xist RNA from two different loci. One expresses the full 17-kb Xist RNA, while the other site expresses a transgenic version, where the Xist 3 end is replaced by a LacZ sequence. Using DNA probes specific for the 3 end of Xist (Cy3, red channel) and LacZ (FITC, green channel), both Xist transcripts can be visualized simultaneously in the cells. DNA is stained with DAPI (blue channel). Previous SectionNext SectionMATERIALSWith the aim of detecting RNA, care must be taken to avoid sample degradation. All glassware should be autoclaved and plasticware should be either certified RNase-free or treated with DEPC-treated H2O and autoclaved.ReagentsCot-1 DNA (Invitrogen)CSK buffer CSK buffer containing 0.5% Triton X-100 Cultured cells for analysis (adherent or from suspension culture)Cy3-dCTP (Amersham Biosciences)Cy3-dCTP should be kept at -20C. Thaw briefly before use.Cy5-dCTP (Amersham Biosciences)Cy5-dCTP should be kept at -20C. Thaw briefly before use.DAPI counterstaining solution DEPC-treated H2O (1% DEPC, Sigma D5758, in deionized H2O) Ethanol (70%, 80%, 95%, and 100%)Formamide (Fluka) Paraformaldehyde solution (4%) for RNA FISH, freshly prepared PBS (1X) PCR clean-up kit (Qiagen)Prime-It II random primer labeling kit (Stratagene; contains exonuclease-free Klenow, 9-mer random primers, and reaction buffer) (for Cy3 or C5 labeling; see Steps 8.i-8.vii) Prime-It Fluor fluorescence labeling kit (Stratagene; contains exonuclease-free Klenow, 9-mer random primers, fluor-12-dUTP, and 5X nucleotide buffer) (for fluorescein labeling; see Steps 8.viii-8.xv) Probe DNARandom priming of 1 g of DNA prepared from plasmids, cosmids, or BAC clones is sufficient for at least 20 hybridizations. It is important that high-quality DNA be used for probe generation. We routinely use Qiagen plasmid purification systems, which give sufficient yield and purity.RNA FISH hybridization mix Salmon sperm DNA (GIBCO)Sodium acetate (NaOAc) (3 M, pH 5.2) SSC (1X, 2X, and 4X; pH 7.4) Vanadyl ribonucleoside complex (200 mM) (Invitrogen) (optional; see Step 4)EquipmentCentrifugeCoplin jarCoverslipsCytospin cartridge (for preparing slides using cytospin technique; see Steps 1.i-1.iv)Humidified chamber, light-protected, 37CNail polishRoboz slides (CellPoint Scientific) (for growing cells on glass slides; see Step 1.v)Roboz slides are commercially available multiwell glass slides. They have been treated for the attachment of mammalian cells and are suitable for growing a large number of cell types including mouse ES cells.Rubber cementSlides (e.g., Superfrost Plus; Sigma)Vectashield mounting medium (Vector Laboratories)Previous SectionNext SectionMETHODBefore getting started, it is important to establish an RNase-free work environment. Work surfaces must be cleaned thoroughly and wiped with ethanol.Slide PreparationAdherent cells can be grown directly onto coverslips. Suspension cells can be placed onto a multiwell slide (Steps 1.v-1.vi) or cytospun onto glass slides (Steps 1.i-1.iv). The cytospin procedure flattens rounded cells, which is not ideal if cell integrity is critical in the experiment. 1. Prepare slides using one of the following methods: To cytospin cells:o i. Prepare a single cell suspension according to standard cell culture protocols. o ii. Add 100 l of the cell suspension (105 cells/ml) to the assembled cytospin cartridge. o iii. Centrifuge the cartridge at room temperature for 5 minutes at 1000 rpm. o iv. The slides are ready for cytoplasmic extraction (Step 2) or can be fixed immediately (Step 6). To grow cells on multiwell glass slides:o v. Place the cells onto a Roboz slide at the proper cell density for 24 hours. o vi. Cells can be fixed directly (Step 6) or after cytoplasmic extraction (Step 2). Cytoplasmic Extraction and FixationIn order to detect nuclear transcripts with a clear fluorescent signal during RNA FISH, the nucleus must be sufficiently permeabilized to allow the probe to gain access to its target. The cells are then fixed to preserve labile RNAs and the integrity of nuclear structure. Some cell types contain an extensive cytoplasm, which has to be extracted in detergent prior to fixation. Since RNA is prone to degradation during the permeabilization steps prior to fixation, the length of the pre-fixation steps should be kept to a minimum. The method below is optimized for detecting nuclear RNA in mouse ES cells and also works for monolayer cells (such as fibroblasts, epithelial cells, or skeletal muscle). 2. Rinse the slides in 1X PBS in a glass Coplin jar. 3. Incubate the slides for 30 seconds in CSK buffer at room temperature. 4. Incubate the slides for 30 seconds to 10 minutes in CSK buffer containing 0.5% Triton X-100 at room temperature. The length of time for detergent extraction depends on the cell type. Because RNA is vulnerable to degradation at this step, vanadyl ribonucleoside complex (10 mM final concentration) can be included in the extraction buffer. 5. Incubate the slides for 30 seconds in CSK buffer at room temperature. 6. Fix the slides in freshly prepared 4% paraformaldehyde for 10 minutes at room temperature. 7. Rinse the slides in 70% ethanol. Slides can be stored in 70% ethanol at 4C until ready to use. Prolonged storage may cause RNA degradation.Random Primer Direct Probe LabelingWe have adapted our labeling protocol from commercially available random primer kits, which generates specific probes with high sensitivity. Fluorescent dye-coupled nucleotides are incorporated directly using exonuclease-free Klenow to generate small probe fragments of 200 nucleotides in length. Longer probe fragments tend to self-associate and form aggregates during the hybridization reaction, which can cause significant background problems. Random priming of 1 g DNA prepared from plasmids, cosmids, or BAC clones serves for at least 20 hybridizations. 8. Prepare the probe using Cy3, Cy5, or fluorescein (FITC). The Cy3/Cy5 protocol (Steps 8.i-8.vii) is based on and uses the reagents from the Prime-It II random primer labeling kit (Stratagene) and Cy3- or Cy5-dCTP from Amersham Biosciences. The fluorescein protocol (Steps 8.viii-8.xv) uses the reagents from Prime-It Fluor fluorescence labeling kit (Stratagene).Probe preparation using Cy3 or Cy5:o i. Denature 1 g of DNA in 23 l of H2O at 95C for 5 minutes. o ii. Add 10 l of 9-mer random primer (provided in the kit). o iii. Denature the reaction mix at 95C for 5 minutes. o iv. Allow the reaction to cool on ice for 5 minutes. o v. Add 10 l of dCTP buffer, 0.5 l of Cy3 or Cy5-dCTP, 1 l (5 U) of exonuclease-free Klenow, and make up to 50 l total volume with H2O. o vi. Incubate at 37C overnight (protected from light). o vii. Continue with probe processing and purification (Step 9). Probe preparation using fluorescein (FITC):o viii. Prepare the reaction buffer by mixing 8 l of fluor-12 dUTP nucleotide and 92 l of 5X nucleotide buffer (provided in the kit). o ix. Denature 1 g of DNA in 23 l of H2O at 95C for 5 minutes. o x. Add 10 l of 9-mer random primer. o xi. Denature the reaction mix at 95C for 5 minutes. o xii. Allow the reaction to cool on ice for 5 minutes. o xiii. Add 10 l reaction buffer to 1 l (5U) of exonuclease-free Klenow and make up to 50 l total volume with H2O. o xiv. Incubate at 37C overnight (protected from light). o xv. Continue with probe processing and purification (Step 9). Post-labeling Probe Processing and Purification 9. Use a PCR clean-up kit to remove unused oligonucleotides. 10. Add 20 l Cot-1 DNA and 10 l salmon sperm DNA to compete for repetitive elements. 11. Precipitate the probe with 1/10 volume 3 M sodium acetate (NaOAc; pH 5.2), 2.5 volumes 100 % ethanol at -80C for 30 minutes. 12. Centrifuge the sample at 4C for 20 minutes. 13. Wash the pellet with 70% ethanol. 14. Resuspend the pellet in 80 l RNA FISH hybridization mix. The probe concentration should be adjusted after a titration on test slides to give a final concentration of 2X. This allows simultaneous hybridization by mixing two probes (e.g., red and green) in a 1:1 ratio at 1X concentration for each probe. Probes used at concentrations that are too high may result in nonspecific background. If the probe is diluted, the sensitivity also decreases. Therefore it is important to determine the optimal probe concentration for each batch of probe generated. The probe can be stored at -20C for at least a year.Hybridization to RNA 15. Denature the probe at 74C for 10 minutes, and let the probe anneal at 37C for 30 minutes. 16. Meanwhile, dehydrate cells on slides through an ethanol series (2 min each in 70%, 80%, 95%, 100%), and then air-dry. 17. Apply 5 l of probe to the cells. 18. Place a coverslip over the probe and seal with rubber cement. 19. Incubate the slide