Chapter 13 Combined RNA/DNA Fluorescence In Situ Hybridization on Whole-Mount Drosophila Ovaries Sergey Shpiz, Sergey Lavrov, and Alla Kalmykova Abstract DNA FISH (fluorescent in situ hybridization) analysis reveals the chromosomal location of the gene of interest. RNA in situ hybridization is used to examine the amounts and cell location of transcripts. This method is commonly used to describe the localization of processed transcripts in different tissues or cell lines. Gene activation studies are often aimed at determining the mechanism of this activation (transcriptional or posttranscriptional). Elucidation of the mechanism of piRNA-mediated silencing of genomic repeats is at the cutting edge of small RNA research. The RNA/DNA FISH technique is a powerful method for assessing transcriptional changes at any particular genomic locus. Colocalization of the RNA and DNA FISH signals allows a determination of the accumulation of nascent transcripts at the transcribed genomic locus. This would be suggest that this gene is activated at the transcriptional (or co-transcriptional) level. Moreover, this method allows for the identification of transcriptional derepression of a distinct copy (copies) among a genomic repeat family. Here, a RNA/DNA FISH protocol is presented for the simultaneous detection of RNA and DNA in situ on whole-mount Drosophila ovaries using tyramide signal amplification. With subsequent immunostaining of chromatin components, this protocol can be easily extended for studying the interdependence between chromatin changes at genomic loci and their transcriptional activity. Key words FISH, Transposable elements, Drosophila, Ovaries, Transcription, TSA, piRNAs
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Introduction Expression of transposable elements (TEs) in Drosophila germline cells is suppressed by piRNAs (PIWI-interacting RNAs) [1, 2]. Disruption of the piRNA machinery leads to activation of transposable element expression. Most likely, both posttranscriptional and transcriptional pathways may be involved in the piRNAmediated silencing of the same target gene. TEs are repressed via piRNA assistance, at least partly, at the transcriptional level [3, 4]. Accumulation of nascent transcripts may be visualized in the proximity of the activated genomic loci by RNA/DNA fluorescent in situ hybridization (FISH, [3, 5, 6]). Expression of different copies
Mikiko C. Siomi (ed.), PIWI-Interacting RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 1093, DOI 10.1007/978-1-62703-694-8_13, © Springer Science+Business Media, LLC 2014
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Fig. 1 Telomeric retroelement TART antisense transcripts accumulate in telomeric regions of the nurse cell nuclei in homozygous spn-E mutants. Antisense TART transcripts (green) are colocalized with the HeT-A DNA probe (red), which serves here as a telomere marker. Thus, these data show the accumulation of nascent TART transcripts at the site of transcription
of the same multiple copy TE family may be regulated in different ways. The position of a particular TE genomic copy may be marked using a DNA FISH probe specific to the unique genomic regions flanking this copy. Such an approach may be useful to detect transcript accumulation for the exact TE copy (or a local set of TE copies). Subsequent chromatin protein immunostaining of the same sample can reveal a correlation between the transcriptional activity of a particular TE copy and the chromatin status of the corresponding genomic locus. Drosophila ovaries are a mixture of somatic and germinal cells with different gene expression profiles. Conventional techniques, like RT-PCR or chromatin immunoprecipitation (ChIP), cannot be easily applied to study gene expression changes on a per-cell basis in such heterogeneous tissues like ovaries. Moreover, knowledge about the expression of a particular copy of TE family in a specific cell type may be very informative. This can be achieved using the DNA/RNA FISH technique. Here, we describe a protocol in which RNA in situ hybridization with a single-stranded riboprobe is performed first because RNA is less stable in comparison with DNA. Next, DNA FISH is done according to standard protocols [7]. The procedure of DNA FISH may impair the level of RNA FISH signals, so we use tyramide signal amplification (TSA, [8, 9]) to preserve RNA signals in the combined DNA/RNA FISH protocol. We applied this technique to describe the transcription of the telomeric TEs HeT-A and TART in the ovaries of the piRNA pathway mutants [3]. TE transcripts are undetectable in wild-type and heterozygous mutant flies. However, we observed an accumulation of telomeric retrotransposon nascent transcripts at the telomeres in homozygous spn-E mutant flies; this suggests the transcriptional activation of the telomeric retrotransposons in the piRNA mutants (Fig. 1).
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Materials 1. 1× PBS (phosphate-buffered saline) (pH 7.4): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, adjust to pH 7.4 with HCl. 2. 1× PBT: 1× PBS containing 0.1 % Tween 20. 3. 3.7 % formaldehyde in 1× PBT. 4. 20× SSC (saline-sodium citrate): 3 M NaCl, 0.3 M Na-citrate, adjust to pH 7.0 with HCl. 5. Hybridization buffer: 50 % formamide, 5× SSC, 0.1 % Tween 20, 100 μg/ml denatured DNA (from herring sperm), 50 μg/ml heparin. 6. Triton X-100. 7. Ultrapure BSA (Ambion). 8. Fluorophore-conjugated tyramide. 9. 3 % H2O2. 10. Anti-DIG-POD antibodies (Roche). 11. Fluorophore-conjugated streptavidin. 12. Normal goat serum (NGS). 13. Formamide deionized. 14. CHAPS (Sigma). 15. BioNick™ Labeling System (Invitrogen). 16. DIG RNA-labeling mix (Roche). 17. TranscriptAid kit (Fermentas). 18. Bio-Rad Micro Bio-Spin 30 column. 19. Image-iT FX (Invitrogen). 20. Slow Fade Gold (Invitrogen).
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Methods The key point of this protocol is tyramide signal amplification (TSA). The mechanism of TSA is shown in Fig. 2. TSA is an enzymemediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP). In the first step, DIG or a Biotinlabeled probe binds via hybridization followed by secondary detection of the probe with an HRP-conjugated antibody or streptavidin conjugate, respectively. Next, HRP activates multiple copies of dye-labeled tyramide in the presence of hydrogen peroxide resulting in highly reactive, short-lived tyramide radicals covalently coupled to tyrosine residues in the proximity of the HRP-target interaction site. Some TSA parameters were optimized.
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Fig. 2 Mechanism of tyramide signal amplification (TSA)
Tyramide concentration. A high tyramide concentration accelerates its binding but leads to higher nonspecific backgrounds and to consumption of the reagent. We searched for an optimal tyramide concentration within a range of 0.2–8 μg/ml and found it to be 4 μg/ml. Peroxide concentration. Hydrogen peroxide concentration determines the rates of tyramide binding and peroxidase inactivation. In a range of 0.001–0.02 %, we found a 0.012 % concentration of peroxide to be optimal. Duration of amplification step. The level of signal amplification depends on the time of reaction: the longer the incubation, the more fluorescent molecules are bound in the hybridization zone. Signal amplification rate is limited by two factors, a gradual increase in background levels and inactivation of probe-bound peroxidase (partly via binding to tyramide). According to previously published protocols, incubation with tyramide varied from 5 up to 45 min. In a range of 15–90 min, we found that 30–60 min was optimal (depending on the intensity of the primary signal). Dye type. Combinations of different RNA-labeling methods and detection methods have been compared: (1) DIG-labeled probe— DIG antibody conjugated with peroxidase [anti-DIG-POD (Roche 11207733910)]; (2) biotinylated probe—avidin conjugated with
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peroxidase [avidin-HRP conjugate (Sigma A7419)]. In both cases, an optimal peroxidase conjugate dilution was found to be 1:500. However, when we used biotinylated probes, background signals were higher than in the case of DIG probes. Biotinylated probes appear to be useful for the detection of mRNA of highly expressed genes. In these cases, the hybridization signal is visible even with high background levels. Generally, we used DIG-labeled RNA probes. 3.1 Preparation of Probes
1. Riboprobe preparation: Carry out in vitro transcription (IVT) using DIG RNA labelling mix (Roche) and the TranscriptAid kit (Fermentas) or its analogues according to the manufacturer’s instructions. PCR-amplified fragments containing the T7 RNA polymerase promoter, or DNA fragments cloned in plasmids containing RNA polymerase promoters, may be used as templates for the synthesis of riboprobes. Synthesized RNA is purified using Bio-Rad Micro Bio-Spin 30 columns according to manufacturer’s specifications. Dry out eluates in a Speedvac up to ~10 μl, and then add 1 ml of hybridization buffer containing 2 μg/μl of yeast RNA. The final concentration of DIG-labeled RNA in this solution is ~10 ng/μl. Alternatively, RNA obtained in the IVT reaction may be precipitated using 5 M ammonium acetate and ethanol. The RNA probe stock solution can be stored at −20 °C for 6 months. 2. DNA probe preparation: Use the BioNick™ Labeling System (Invitrogen) according to manufacturer’s instructions to prepare the biotin-labeled probes. Dissolve labeled probe in 80 μl of hybridization buffer containing 2 μg/μl of yeast RNA. The final concentration of biotinylated probe in the stock solution is ~25 ng/μl. The DNA probe stock solution can be stored at −20 °C for 1 year.
3.2
RNA FISH
1. Dissect the ovaries from ~50 Drosophila females in PBT and store in a 1.5-ml Eppendorf tube on ice during isolation (up to 2 h). For RNA FISH, it is important to preserve organ integrity at this step. 2. Fix ovaries for 20 min in 3.7 % formaldehyde, 1× PBT at room temperature (RT) on a rotating wheel. 3. Wash ovaries with PBT for 5 min three times on a rotating wheel. After this step, tissues can be stored at 4 °C overnight. It is recommended to disunite the ovarioles with needles or tweezers (see Note 1). 4. Incubate ovaries in 0.6 % Triton X-100, 1× PBS for 10 min at RT on a rotating wheel (see Note 2). 5. Wash ovaries with PBT for 5 min three times on a rotating wheel. After this step, tissues can be stored at 4 °C overnight.
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6. Prepare fresh solutions of 20, 50, and 80 % hybridization buffer in PBT. Transfer samples stepwise to hybridization buffer by incubation in 1 ml of each solution for 15 min. 7. Incubate samples in 100 % hybridization buffer for 15 min. 8. Preincubate ovaries in hybridization buffer at 42 °C for 1–3 h. 9. Denature 100 μl of DIG-labeled riboprobe solution from step 1 in Subheading 3.1 at 80 °C for 10 min. 10. After removing the supernatant from step 8, add 100 μl of denaturated DIG-labeled riboprobe solution from step 9. Overlay the sample with mineral oil. 11. Incubate at 42 °C overnight with shaking (see Note 3). 12. Wash two times with 1 ml of 50 % formamide, 2× SSC, 0.3 % CHAPS for 15 min at 60 °C with shaking. 13. Wash with 1 ml of 40 % formamide, 2× SSC, 0.3 % CHAPS for 15 min at 42 °C with shaking. 14. Incubate sequentially for 15 min at 42 °C with shaking in 1 ml of the following solutions: (a) 1× PBT, 30 % formamide (b) 1× PBT, 20 % formamide (c) 1× PBT, 10 % formamide 15. Wash ovaries with PBT for 5 min three times at RT. 16. Block in PBT containing 5 % NGS, 1 % BSA, 0.3 % Triton X-100 for 1–3 h. 17. Optional. Incubate with 1 % H2O2 in PBT for 30 min at RT to inactivate endogenous peroxidase (see Note 4). 18. Incubate with anti-DIG POD antibodies (Roche) (diluted at 1:500) in PBT, 0.3 % Triton X-100, 3 % NGS for at least 1 h on a rotating wheel at RT. 19. Wash ovaries with PBT for 10 min five times at RT. 20. Incubate with fluorophore-conjugated tyramide (4 μg/ml) in PBT for 30 min. 21. Add H2O2 to a final concentration of 0.012 % and incubate for 30–60 min at RT with rotation. 22. Wash ovaries with PBT for 5 min three times at RT. 23. Check RNA signal under fluorescent microscope (see Note 5). Tissues can be stored at 4 °C for several days after this step. 3.3
DNA FISH
Take tissue samples from step 22 in Subheading 3.2. 1. Optional. Add RNAse A to a final concentration of 100 μg/ml. Incubate for 4 h at 37 °C or overnight at 4 °C. 2. Wash ovaries with PBT for 5 min three times at RT.
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3. Prepare fresh solutions of 20, 50, and 80 % hybridization buffer in PBT. Incubate samples for 15 min in 1 ml of each solution. 4. Incubate in 100 % hybridization buffer for 15 min to replace the PBT with hybridization buffer. 5. After removing the supernatant from the last step, add 20 μl of biotin-labeled probe solution from step 2 in Subheading 3.1. If you use biotinylated oligonucleotides as a probe, their concentration in hybridization solution should be 0.5 pM/μl. Overlay with mineral oil. 6. Incubate ovaries at 80 °C for 15 min to denature DNA. 7. Hybridize with probe overnight at 37 °C (see Note 6) with shaking (see Note 3). 8. Wash two times with 1 ml of 50 % formamide, 2× SSC, 0.3 % CHAPS for 15 min at 37 °C with shaking. 9. Wash with 1 ml of 40 % formamide, 2× SSC, 0.3 % CHAPS for 15 min at 37 °C with shaking. 10. Incubate sequentially for 15 min at 37 °C with shaking in 1 ml of the following solutions: (a) 1× PBT, 30 % formamide (b) 1× PBT, 20 % formamide (c) 1× PBT, 10 % formamide 11. Wash ovaries with PBT for 5 min three times at RT. Tissues can be stored at 4 °C for several days after this step. 3.4 DNA Staining and Immunostaining
Take tissue samples from step 11 in Subheading 3.3. 1. Replace PBT with Image-iT FX (Invitrogen) and incubate for 30 min at RT (see Note 7). 2. Wash ovaries with PBT for 5 min three times at RT. 3. Optional: Block in PBT containing 5 % NGS, 1 % BSA, 0.3 % Triton X-100 for 1–3 h. 4. Incubate with primary antibodies specific for the protein of interest in PBT containing 3 % NGS for 1 h on a rotating wheel. 5. Wash ovaries with PBT for 5 min three times at RT. 6. Incubate with fluorophore-conjugated streptavidin and fluorophore-conjugated secondary antibodies in PBT, 0.3 % Triton X-100, 3 % NGS for 1 h on a rotating wheel (see Note 7). 7. Wash ovaries with PBT, 0.3 % Triton X-100 for 5 min three times at RT. 8. Incubate in 5 μM DAPI in PBT for 10 min at RT. 9. Wash ovaries in PBT for 5 min three times at RT. 10. Remove supernatant and add 60 μl of Slow Fade Gold solution (Invitrogen).
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11. Pipette ovaries onto a slide. Lower a coverslip onto the tissues. Seal the coverslip to the slide with nail polish. 12. Analyze samples by conventional fluorescence or confocal microscopy.
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Notes 1. The disjunction of distinct ovarioles is recommended at this step to enhance the accessibility of ovarian cells by the labeled probes and antibodies. 2. Incubation in 0.6 % Triton X-100 is used to improve subsequent probe and antibody penetration. This step can be replaced by the following procedures: treatment with 50 μg/ml proteinase K solution for 2–15 min (should be adjusted); incubation in 0.3 % Triton X-100 with 0.3 % sodium deoxycholate in PBS twice for 30 min; freezing/thawing. 3. Step 11 in Subheading 3.2, steps 6 and 7 in Subheading 3.3, as well as washing steps, may be performed in an Eppendorf Thermo mixer at 850 rpm shaking. All solutions used in these steps should be preheated to the appropriate temperature. 4. The activity of endogenous peroxidases may result in a high level of background; therefore, it may be useful to inactivate them with a high concentration of hydrogen peroxide before binding. However, in formaldehyde-fixed samples, endogenous peroxidases are inactivated. We have not pretreated samples with hydrogen peroxide. 5. Hybridization conditions applied in Subheading 3.2 allowed us to detect RNA rather than genomic DNA. To exclude the possibility of RNA/DNA hybridization at this stage, an RNase H control may be done. After hybridization and washing (after step 15 in Subheading 3.2), incubate ovaries in PBS containing 0.3 u/μl RNase H for 1 h at 37 °C. 6. If biotinylated oligonucleotides are used, the hybridization temperature should be adjusted taking into account the length and melting temperature of the oligonucleotide. Commonly, 30 °C is a suitable hybridization temperature. 7. Background staining emerging from fluorescent reagents is largely eliminated when the Image-iT™ FX signal enhancer is applied to fixed and permeabilized cells prior to staining. It may occur that, in the course of immunostaining with particular antibody combinations, incubation with Image-iT FX reduces signal-to-noise ratios; it is recommended that you check if it occurs in your specific case. If so, skip steps 1 and 2 in Subheading 3.4, but perform step 3 in Subheading 3.4 for blocking.
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Acknowledgments This work was supported by grants to S.S. from the Russian Foundation for Basic Researches (12-04-00996) and to A.K. from the Russian Academy of Sciences Program for Molecular and Cell Biology. References 1. Aravin AA, Hannon GJ, Brennecke J (2007) The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318:761–764 2. Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD (2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313:320–324 3. Shpiz S, Olovnikov I, Sergeeva A, Lavrov S, Abramov Y, Savitsky M, Kalmykova A (2011) Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons. Nucleic Acids Res 39:8703–8711 4. Klenov MS, Lavrov SA, Stolyarenko AD, Ryazansky SS, Aravin AA, Tuschl T, Gvozdev VA (2007) Repeat-associated siRNAs cause chromatin silencing of retrotransposons in the Drosophila melanogaster germline. Nucleic Acids Res 35:5430–5438
5. Takizawa T, Gudla PR, Guo L, Lockett S, Misteli T (2008) Allele-specific nuclear positioning of the monoallelically expressed astrocyte marker GFAP. Genes Dev 22:489–498 6. Shpiz S, Kwon D, Rozovsky Y, Kalmykova A (2009) rasiRNA pathway controls antisense expression of Drosophila telomeric retrotransposons in the nucleus. Nucleic Acids Res 37: 268–278 7. Lavrov S, Dejardin J, Cavalli G (2004) Combined immunostaining and FISH analysis of polytene chromosomes. Methods Mol Biol 247:289–303 8. Speel EJ, Komminoth P (1999) CARD in situ hybridization: sights and signals. Endocr Pathol 10:193–198 9. Speel EJ, Hopman AH, Komminoth P (1999) Amplification methods to increase the sensitivity of in situ hybridization: play card(s). J Histochem Cytochem 47:281–288
1
Introduction Expression of transposable elements (TEs) in Drosophila germline cells is suppressed by piRNAs (PIWI-interacting RNAs) [1, 2]. Disruption of the piRNA machinery leads to activation of transposable element expression. Most likely, both posttranscriptional and transcriptional pathways may be involved in the piRNAmediated silencing of the same target gene. TEs are repressed via piRNA assistance, at least partly, at the transcriptional level [3, 4]. Accumulation of nascent transcripts may be visualized in the proximity of the activated genomic loci by RNA/DNA fluorescent in situ hybridization (FISH, [3, 5, 6]). Expression of different copies
Mikiko C. Siomi (ed.), PIWI-Interacting RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 1093, DOI 10.1007/978-1-62703-694-8_13, © Springer Science+Business Media, LLC 2014
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Fig. 1 Telomeric retroelement TART antisense transcripts accumulate in telomeric regions of the nurse cell nuclei in homozygous spn-E mutants. Antisense TART transcripts (green) are colocalized with the HeT-A DNA probe (red), which serves here as a telomere marker. Thus, these data show the accumulation of nascent TART transcripts at the site of transcription
of the same multiple copy TE family may be regulated in different ways. The position of a particular TE genomic copy may be marked using a DNA FISH probe specific to the unique genomic regions flanking this copy. Such an approach may be useful to detect transcript accumulation for the exact TE copy (or a local set of TE copies). Subsequent chromatin protein immunostaining of the same sample can reveal a correlation between the transcriptional activity of a particular TE copy and the chromatin status of the corresponding genomic locus. Drosophila ovaries are a mixture of somatic and germinal cells with different gene expression profiles. Conventional techniques, like RT-PCR or chromatin immunoprecipitation (ChIP), cannot be easily applied to study gene expression changes on a per-cell basis in such heterogeneous tissues like ovaries. Moreover, knowledge about the expression of a particular copy of TE family in a specific cell type may be very informative. This can be achieved using the DNA/RNA FISH technique. Here, we describe a protocol in which RNA in situ hybridization with a single-stranded riboprobe is performed first because RNA is less stable in comparison with DNA. Next, DNA FISH is done according to standard protocols [7]. The procedure of DNA FISH may impair the level of RNA FISH signals, so we use tyramide signal amplification (TSA, [8, 9]) to preserve RNA signals in the combined DNA/RNA FISH protocol. We applied this technique to describe the transcription of the telomeric TEs HeT-A and TART in the ovaries of the piRNA pathway mutants [3]. TE transcripts are undetectable in wild-type and heterozygous mutant flies. However, we observed an accumulation of telomeric retrotransposon nascent transcripts at the telomeres in homozygous spn-E mutant flies; this suggests the transcriptional activation of the telomeric retrotransposons in the piRNA mutants (Fig. 1).
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Materials 1. 1× PBS (phosphate-buffered saline) (pH 7.4): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, adjust to pH 7.4 with HCl. 2. 1× PBT: 1× PBS containing 0.1 % Tween 20. 3. 3.7 % formaldehyde in 1× PBT. 4. 20× SSC (saline-sodium citrate): 3 M NaCl, 0.3 M Na-citrate, adjust to pH 7.0 with HCl. 5. Hybridization buffer: 50 % formamide, 5× SSC, 0.1 % Tween 20, 100 μg/ml denatured DNA (from herring sperm), 50 μg/ml heparin. 6. Triton X-100. 7. Ultrapure BSA (Ambion). 8. Fluorophore-conjugated tyramide. 9. 3 % H2O2. 10. Anti-DIG-POD antibodies (Roche). 11. Fluorophore-conjugated streptavidin. 12. Normal goat serum (NGS). 13. Formamide deionized. 14. CHAPS (Sigma). 15. BioNick™ Labeling System (Invitrogen). 16. DIG RNA-labeling mix (Roche). 17. TranscriptAid kit (Fermentas). 18. Bio-Rad Micro Bio-Spin 30 column. 19. Image-iT FX (Invitrogen). 20. Slow Fade Gold (Invitrogen).
3
Methods The key point of this protocol is tyramide signal amplification (TSA). The mechanism of TSA is shown in Fig. 2. TSA is an enzymemediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP). In the first step, DIG or a Biotinlabeled probe binds via hybridization followed by secondary detection of the probe with an HRP-conjugated antibody or streptavidin conjugate, respectively. Next, HRP activates multiple copies of dye-labeled tyramide in the presence of hydrogen peroxide resulting in highly reactive, short-lived tyramide radicals covalently coupled to tyrosine residues in the proximity of the HRP-target interaction site. Some TSA parameters were optimized.
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Fig. 2 Mechanism of tyramide signal amplification (TSA)
Tyramide concentration. A high tyramide concentration accelerates its binding but leads to higher nonspecific backgrounds and to consumption of the reagent. We searched for an optimal tyramide concentration within a range of 0.2–8 μg/ml and found it to be 4 μg/ml. Peroxide concentration. Hydrogen peroxide concentration determines the rates of tyramide binding and peroxidase inactivation. In a range of 0.001–0.02 %, we found a 0.012 % concentration of peroxide to be optimal. Duration of amplification step. The level of signal amplification depends on the time of reaction: the longer the incubation, the more fluorescent molecules are bound in the hybridization zone. Signal amplification rate is limited by two factors, a gradual increase in background levels and inactivation of probe-bound peroxidase (partly via binding to tyramide). According to previously published protocols, incubation with tyramide varied from 5 up to 45 min. In a range of 15–90 min, we found that 30–60 min was optimal (depending on the intensity of the primary signal). Dye type. Combinations of different RNA-labeling methods and detection methods have been compared: (1) DIG-labeled probe— DIG antibody conjugated with peroxidase [anti-DIG-POD (Roche 11207733910)]; (2) biotinylated probe—avidin conjugated with
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peroxidase [avidin-HRP conjugate (Sigma A7419)]. In both cases, an optimal peroxidase conjugate dilution was found to be 1:500. However, when we used biotinylated probes, background signals were higher than in the case of DIG probes. Biotinylated probes appear to be useful for the detection of mRNA of highly expressed genes. In these cases, the hybridization signal is visible even with high background levels. Generally, we used DIG-labeled RNA probes. 3.1 Preparation of Probes
1. Riboprobe preparation: Carry out in vitro transcription (IVT) using DIG RNA labelling mix (Roche) and the TranscriptAid kit (Fermentas) or its analogues according to the manufacturer’s instructions. PCR-amplified fragments containing the T7 RNA polymerase promoter, or DNA fragments cloned in plasmids containing RNA polymerase promoters, may be used as templates for the synthesis of riboprobes. Synthesized RNA is purified using Bio-Rad Micro Bio-Spin 30 columns according to manufacturer’s specifications. Dry out eluates in a Speedvac up to ~10 μl, and then add 1 ml of hybridization buffer containing 2 μg/μl of yeast RNA. The final concentration of DIG-labeled RNA in this solution is ~10 ng/μl. Alternatively, RNA obtained in the IVT reaction may be precipitated using 5 M ammonium acetate and ethanol. The RNA probe stock solution can be stored at −20 °C for 6 months. 2. DNA probe preparation: Use the BioNick™ Labeling System (Invitrogen) according to manufacturer’s instructions to prepare the biotin-labeled probes. Dissolve labeled probe in 80 μl of hybridization buffer containing 2 μg/μl of yeast RNA. The final concentration of biotinylated probe in the stock solution is ~25 ng/μl. The DNA probe stock solution can be stored at −20 °C for 1 year.
3.2
RNA FISH
1. Dissect the ovaries from ~50 Drosophila females in PBT and store in a 1.5-ml Eppendorf tube on ice during isolation (up to 2 h). For RNA FISH, it is important to preserve organ integrity at this step. 2. Fix ovaries for 20 min in 3.7 % formaldehyde, 1× PBT at room temperature (RT) on a rotating wheel. 3. Wash ovaries with PBT for 5 min three times on a rotating wheel. After this step, tissues can be stored at 4 °C overnight. It is recommended to disunite the ovarioles with needles or tweezers (see Note 1). 4. Incubate ovaries in 0.6 % Triton X-100, 1× PBS for 10 min at RT on a rotating wheel (see Note 2). 5. Wash ovaries with PBT for 5 min three times on a rotating wheel. After this step, tissues can be stored at 4 °C overnight.
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6. Prepare fresh solutions of 20, 50, and 80 % hybridization buffer in PBT. Transfer samples stepwise to hybridization buffer by incubation in 1 ml of each solution for 15 min. 7. Incubate samples in 100 % hybridization buffer for 15 min. 8. Preincubate ovaries in hybridization buffer at 42 °C for 1–3 h. 9. Denature 100 μl of DIG-labeled riboprobe solution from step 1 in Subheading 3.1 at 80 °C for 10 min. 10. After removing the supernatant from step 8, add 100 μl of denaturated DIG-labeled riboprobe solution from step 9. Overlay the sample with mineral oil. 11. Incubate at 42 °C overnight with shaking (see Note 3). 12. Wash two times with 1 ml of 50 % formamide, 2× SSC, 0.3 % CHAPS for 15 min at 60 °C with shaking. 13. Wash with 1 ml of 40 % formamide, 2× SSC, 0.3 % CHAPS for 15 min at 42 °C with shaking. 14. Incubate sequentially for 15 min at 42 °C with shaking in 1 ml of the following solutions: (a) 1× PBT, 30 % formamide (b) 1× PBT, 20 % formamide (c) 1× PBT, 10 % formamide 15. Wash ovaries with PBT for 5 min three times at RT. 16. Block in PBT containing 5 % NGS, 1 % BSA, 0.3 % Triton X-100 for 1–3 h. 17. Optional. Incubate with 1 % H2O2 in PBT for 30 min at RT to inactivate endogenous peroxidase (see Note 4). 18. Incubate with anti-DIG POD antibodies (Roche) (diluted at 1:500) in PBT, 0.3 % Triton X-100, 3 % NGS for at least 1 h on a rotating wheel at RT. 19. Wash ovaries with PBT for 10 min five times at RT. 20. Incubate with fluorophore-conjugated tyramide (4 μg/ml) in PBT for 30 min. 21. Add H2O2 to a final concentration of 0.012 % and incubate for 30–60 min at RT with rotation. 22. Wash ovaries with PBT for 5 min three times at RT. 23. Check RNA signal under fluorescent microscope (see Note 5). Tissues can be stored at 4 °C for several days after this step. 3.3
DNA FISH
Take tissue samples from step 22 in Subheading 3.2. 1. Optional. Add RNAse A to a final concentration of 100 μg/ml. Incubate for 4 h at 37 °C or overnight at 4 °C. 2. Wash ovaries with PBT for 5 min three times at RT.
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3. Prepare fresh solutions of 20, 50, and 80 % hybridization buffer in PBT. Incubate samples for 15 min in 1 ml of each solution. 4. Incubate in 100 % hybridization buffer for 15 min to replace the PBT with hybridization buffer. 5. After removing the supernatant from the last step, add 20 μl of biotin-labeled probe solution from step 2 in Subheading 3.1. If you use biotinylated oligonucleotides as a probe, their concentration in hybridization solution should be 0.5 pM/μl. Overlay with mineral oil. 6. Incubate ovaries at 80 °C for 15 min to denature DNA. 7. Hybridize with probe overnight at 37 °C (see Note 6) with shaking (see Note 3). 8. Wash two times with 1 ml of 50 % formamide, 2× SSC, 0.3 % CHAPS for 15 min at 37 °C with shaking. 9. Wash with 1 ml of 40 % formamide, 2× SSC, 0.3 % CHAPS for 15 min at 37 °C with shaking. 10. Incubate sequentially for 15 min at 37 °C with shaking in 1 ml of the following solutions: (a) 1× PBT, 30 % formamide (b) 1× PBT, 20 % formamide (c) 1× PBT, 10 % formamide 11. Wash ovaries with PBT for 5 min three times at RT. Tissues can be stored at 4 °C for several days after this step. 3.4 DNA Staining and Immunostaining
Take tissue samples from step 11 in Subheading 3.3. 1. Replace PBT with Image-iT FX (Invitrogen) and incubate for 30 min at RT (see Note 7). 2. Wash ovaries with PBT for 5 min three times at RT. 3. Optional: Block in PBT containing 5 % NGS, 1 % BSA, 0.3 % Triton X-100 for 1–3 h. 4. Incubate with primary antibodies specific for the protein of interest in PBT containing 3 % NGS for 1 h on a rotating wheel. 5. Wash ovaries with PBT for 5 min three times at RT. 6. Incubate with fluorophore-conjugated streptavidin and fluorophore-conjugated secondary antibodies in PBT, 0.3 % Triton X-100, 3 % NGS for 1 h on a rotating wheel (see Note 7). 7. Wash ovaries with PBT, 0.3 % Triton X-100 for 5 min three times at RT. 8. Incubate in 5 μM DAPI in PBT for 10 min at RT. 9. Wash ovaries in PBT for 5 min three times at RT. 10. Remove supernatant and add 60 μl of Slow Fade Gold solution (Invitrogen).
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11. Pipette ovaries onto a slide. Lower a coverslip onto the tissues. Seal the coverslip to the slide with nail polish. 12. Analyze samples by conventional fluorescence or confocal microscopy.
4
Notes 1. The disjunction of distinct ovarioles is recommended at this step to enhance the accessibility of ovarian cells by the labeled probes and antibodies. 2. Incubation in 0.6 % Triton X-100 is used to improve subsequent probe and antibody penetration. This step can be replaced by the following procedures: treatment with 50 μg/ml proteinase K solution for 2–15 min (should be adjusted); incubation in 0.3 % Triton X-100 with 0.3 % sodium deoxycholate in PBS twice for 30 min; freezing/thawing. 3. Step 11 in Subheading 3.2, steps 6 and 7 in Subheading 3.3, as well as washing steps, may be performed in an Eppendorf Thermo mixer at 850 rpm shaking. All solutions used in these steps should be preheated to the appropriate temperature. 4. The activity of endogenous peroxidases may result in a high level of background; therefore, it may be useful to inactivate them with a high concentration of hydrogen peroxide before binding. However, in formaldehyde-fixed samples, endogenous peroxidases are inactivated. We have not pretreated samples with hydrogen peroxide. 5. Hybridization conditions applied in Subheading 3.2 allowed us to detect RNA rather than genomic DNA. To exclude the possibility of RNA/DNA hybridization at this stage, an RNase H control may be done. After hybridization and washing (after step 15 in Subheading 3.2), incubate ovaries in PBS containing 0.3 u/μl RNase H for 1 h at 37 °C. 6. If biotinylated oligonucleotides are used, the hybridization temperature should be adjusted taking into account the length and melting temperature of the oligonucleotide. Commonly, 30 °C is a suitable hybridization temperature. 7. Background staining emerging from fluorescent reagents is largely eliminated when the Image-iT™ FX signal enhancer is applied to fixed and permeabilized cells prior to staining. It may occur that, in the course of immunostaining with particular antibody combinations, incubation with Image-iT FX reduces signal-to-noise ratios; it is recommended that you check if it occurs in your specific case. If so, skip steps 1 and 2 in Subheading 3.4, but perform step 3 in Subheading 3.4 for blocking.
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Acknowledgments This work was supported by grants to S.S. from the Russian Foundation for Basic Researches (12-04-00996) and to A.K. from the Russian Academy of Sciences Program for Molecular and Cell Biology. References 1. Aravin AA, Hannon GJ, Brennecke J (2007) The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318:761–764 2. Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD (2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313:320–324 3. Shpiz S, Olovnikov I, Sergeeva A, Lavrov S, Abramov Y, Savitsky M, Kalmykova A (2011) Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons. Nucleic Acids Res 39:8703–8711 4. Klenov MS, Lavrov SA, Stolyarenko AD, Ryazansky SS, Aravin AA, Tuschl T, Gvozdev VA (2007) Repeat-associated siRNAs cause chromatin silencing of retrotransposons in the Drosophila melanogaster germline. Nucleic Acids Res 35:5430–5438
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