Digital Commons@Becker Digital Commons@Becker RNA in situ hybridization of dissected gonads RNA in situ hybridization of dissected gonads

Copyright: © 2006 Min-Ho Lee and Tim Schedl. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. To whom correspondence should be addressed. E-mail: mhlee@albany.edu or ts@genetics.wustl.edu Current address: Department of Biological Sciences, University at Albany, SUNY, Albany, NY 12222 RNA in situ hybridization of dissected gonads* Min-Ho Lee, Tim Schedl, Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110 USA


Introduction
Determination of the temporal and spatial pattern of gene expression is an important step in understanding gene function during C. elegans development. The expression pattern of a gene can be examined in several ways including transgenic reporter fusions (e.g., gfp, β-galactosidase), as well as immunohistochemistry and RNA in situ hybridization for analysis of the endogenous protein and mRNA, respectively. While reporter fusions and immnohistochemistry require time and effort to generate reagents (reporter construction and generation of transgenic lines or antibody production), RNA in situ hybridization only requires the transcribed region of a gene of interest. In addition, as a complement to protein expression patterns of a gene, RNA in situ allows one to determine whether the mRNA is subjected to post-transcriptional controls such as differential RNA localization, stability, and/or translational regulation Seydoux and Fire 1994;Jones et al., 1996;Schedl 2001, 2004).
Several in situ hybridization protocols have been developed to detect RNA in C. elegans. RNA in situ in embryos is well described previously (Seydoux and Fire, 1995. Protocols for whole-mount in situ of intact animals and embryos can be also found at Yuji Kohara's website. Fluorescence RNA in situ hybridizations have been also described (Albertson et al., 1995;Graves, et al., 1999;Pitt et al., 2000). In this chapter, a protocol for detection of mRNA by in situ hybridization to dissected gonads is described. This protocol is primarily based on the method of Seydoux and Fire (1995) as adapted for dissected gonads.
In contrast to an earlier method described for dissected gonads wherein the gonads were affixed to slides , the various manipulations described below (gonad dissections, hybridizations, and washes) are all performed in solution, either in glass dishes (dissection) or in small glass tubes (hybridization). Washes are done by low-speed spins and all solutions (except fixatives) contain 0.1% Tween 20 to prevent dissected gonads from sticking to glass. A key to make this protocol work consistently is to fix dissected gonads in a combination of paraformaldehyde (3%) and glutaraldehyde (0.25%). The addition of glutaraldehyde results in a lower background, as compared to fixation with paraformaldehyde alone and makes gonads both less sticky and less susceptible to breakage.
It is important to perform RNA in situ hybridization using both sense and anti-sense probes for a gene of interest, at least for initial experiments. RNA in situ hybridization with a sense probe should give little or no signal compared to that with an anti-sense probe ( Figure 1). The sense probe background control is particularly important for detecting transcripts that are expressed at very low levels, which requires an extended developing time with the alkaline phosphatase-mediated detection system. In general, we can easily detect most mRNAs that have five or more EST clones reported in WormBase. For mRNAs that have less than two EST clones (therefore likely expressed at very low levels), it is helpful to concentrate the probe either by using more template in the asymmetric PCR (probe synthesis step) or by dissolving the digoxigenin (DIG) labeled probe in a smaller volume. RNA in situ hybridization with a concentrated anti-sense probe usually results in increased signal with decreased developing time of the alkaline phosphatase-mediated detection system. In addition, concentration of the sense probe does not increase the background signal significantly. showing cep-1/p53 mRNA accumulation in wild-type (A) and gld-1(null) (B) hermaphrodites, and wild-type male (C). Each panel has images from gonads hybridized with an anti-sense probe and a sense probe otherwise treated equally. Each image shows a surface view of the gonad where the distal region is on the left and the proximal region is on the right. Sense probe gave no or very little signals (the proximal signal is likely due to endogenous phosphatases). In wild-type hermaphrodites, cep-1 mRNA accumulates throughout the germ line and its levels increase toward the proximal region of the germ line. In gld-1(null), cep-1 mRNA levels are slightly lower as compared with wild type. In wild-type males, cep-1 mRNA accumulates at higher levels only in the distal meiotic germ cells, showing sexual dimorphism.

General expression pattern
The levels of most maternal mRNAs become higher in the middle of the pachytene region and remain high in the developing oocytes in the proximal region because the bulk of RNA synthesis for oogenesis occurs in pachytene stage germ cells, with progressively less RNA synthesis as germ cells proceed through the diplotene stage. As chromosomes become highly condensed in late stage oocytes (diakinesis), almost no de novo RNA synthesis occurs (Gibert et al., 1984;Schisa et al., 2001) and RNA polymerase II becomes inactive (Kelly et al., 2002). Therefore, most maternal mRNAs are transcribed primarily in pachytene stage germ cells in the distal region and transported into the common cytoplasmic core. In the proximal region, maternal mRNAs are packaged into growing oocytes. These processes result in extensive RNA accumulation throughout pachytene stage germ cells and developing oocytes ( Figure 1). Some mRNAs are expressed throughout the germline including the distal mitotic region (e.g., glp-1) while others are only expressed in meiotic cells (e.g., rme-2). For a number of transcripts expressed throughout the germline, the relative levels observed in the distal mitotic region are lower, compared to the levels in the meiotic pachytene cells and proximal oocytes ( Figure 1). The difference is likely, at least in part, a reflection of a lower level of the mRNA/protein being required for function in the distal mitotic region, while a higher level is required for late oogenesis and/or maternally for early embryogenesis.

Materials
Solutions do not need to be treated with diethyl pyrocarbonate.
PBS: Make a 10x stock solution according to Sambrook et al. (1989).
Paraformaldehyde / glutaraldehyde fix:Paraformaldehyde/Glutaraldehyde fixation works well for germline RNA in situ: gonads remain intact through an extensive protease treatment (50 µg/ml Proteinase K for 30 min at room temperature (RT) that would destroy gonads fixed in paraformaldehyde alone. This fixation can also be used with good results for histological staining with some antibodies.
Alkaline-phosphatase-conjugated anti-DIG (Fab2 fragment) from Roche (#1 093 274): An optimal dilution of this antibody needs to be determined empirically as lot-to-lot variations may exist. We have been using dilutions from 1:1000 to 1:2500.
Sigma Fast BCIP/NBT tablet (#B5655): Dissolve one tablet in 10 ml staining solution. If non-specific background signal is high, you may want to use more staining solution (up to 20 ml) to dissolve the tablet.
Anti-Fade solution: Make 0.2M Dabco (Sigma #D2522) in 20 mM Tris (pH 8.0), which can be stored at −20°C. Then dilute this solution (10%) in glycerol (90%) and store −20°C. ‡ Use only one primer. We set up two reactions, one to generate a sense probe with 5'primer and the other to generate an anti-sense probe with 3'primer. RNA in situ hybridization with the sense probe will be a background control.

Procedure
Single-stranded probes from cloned cDNA can be prepared anytime. Following dissections and fixation, fixed gonads can be stored in 100% MeOH at −20°C for at least one week. Once the gonads are subjected to permeabilization, the remaining procedure has to be completed.

Procedure 1. Preparation of single-stranded probes from cloned cDNA
Based on Patel and Goodman (1992), with slight modifications.

PCR amplification of a cDNA of interest with any set of primers.
A cloned cDNA template is optimal because amplified products from genomic DNA usually generate high background.
Use less than 1µl (10 pmole/µl) of primers because QIAquick PCR Purification kit will not effectively remove primers if the concentration is higher.
We usually set up two 50 µl PCR reaction with 35 cycles (94°C 30 seconds, 55°C 30 seconds and 72°C, 1 minute/1 kb). Run 3 µl each in an agarose gel; the PCR product must be an intense single band.

Purification of PCR product
Use the QIAquick PCR Purification kit to purify the PCR product away from unincorporated dNTP and PCR primers. Elute with 25 µl Elution buffer. The concentration should be above 200 ng/µl.
6. The probe is boiled for 1 hr. This step reduces the length of the probe for efficient penetration.
7. Probes can be stored at −20°C for a long period. We have used two year-old probe without any obvious reduction of signal.

Procedure 2. Dissections and fixation
obtained at dilutions between 1:2 and 1:5, but it began to fall off at 1:10. Probe to major sperm protein RNA, which is very abundant during spermatogenesis, could be diluted 1:20 without any fall off in signal.
5. Hybridize for 12 to 36 hr at 48°C (longer hybridization times tend to give increased signal and lower background). We routinely hybridize for 36 hrs.
8. Add 400 µl of HB that is pre-warmed to 48°, spin down, and remove supernatant.
9. Divide gonads among several 6 x 50 mm culture tubes, one tube per hybridzation. 4. Transfer gonads to staining solution with BCIP/NDT and 100 ng/ml DAPI and cover to protect from light. Signal will come up anywhere between 5 min and 6 hr. For convenience, the reaction can be monitored by transferring tube contents to a glass dish and inspecting periodically under the dissecting scope. However, stain will appear darker in the dissecting scope than under Nomarski optics with a 40X or higher lens.
5. Stop the reaction by washing three times in PBTw. Finally place gonads in PBS containing 100 ng/ml DAPI.
6. After removing DAPI solution as much as possible, add 35 µl anti-fade solution.
Procedure 6. Mounting gonads for viewing • Using a drawn capillary pipette, transfer settled worms onto a large 2% agar pad that covers most of a slide. After drawing off excess liquid with a capillary, an eyelash hair can be used to push gonads and intestines away from one another. Cover with a large (24 x 50 mm) coverslip, taking care not to move the coverslip once in place. Also do not seal the coverslip immediately -image may improve as liquid evaporates and gonads become somewhat flattened. We usually take images after storing the slides at 4°C overnight without sealing.
Bright field (Nomarski) images must be taken before DAPI images because the strong UV light focused by the microscope literally burns gonads brown. Slides can be stored at 4°C for a week or more, particularly if sealed with nail polish around the periphery of the coverslip. Because the stain is purplish, images are better in color than in black and white.

Acknowledgements
We are grateful to Ross Francis for establishing this protocol and members of the Schedl lab for comments on this review. Work in the TS lab is supported by NIH grant GM63310.