Fisherbrand unbreakable coverslips (Fisher Scienti fi c, Suwanee,. GA, USA). 1. RNAlater (Qiagen, Hilden, Germany). 2. DNA stain (e.g., DAPI or SYTOX green).
Chapter 20 Analyzing the Meiotic Transcriptome Using Isolated Meiocytes of Arabidopsis thaliana Changbin Chen and Ernest F. Retzel Abstract Improved transcriptome sequencing technologies (RNA-seq) have advanced our understanding of the tissue-specific transcriptome landscapes, including those of messenger RNAs, noncoding RNAs and small RNAs. However, transcriptome profiles of plant meiocytes remain challenging due to the lack of efficient methods to enrich meiocytes for the analysis of temporal and spatial gene expression patterns during meiosis. In this chapter, we describe a method to analyze the Arabidopsis meiotic transcriptome using isolated male meiocytes. Keywords RNA-seq, Meiocytes, Transcriptome, Capillary collection method, Arabidopsis
1
Introduction Sequence-based differential expression (RNA-seq) has largely replaced microarrays for gene expression studies (1–8). The advantages of RNA-seq include the ability to discover novel genes and exons, alternate splicing, and short insertions and deletions (indels). Even in the completely sequenced and well-studied model plant Arabidopsis thaliana, this technique enabled us to discover genes in the meiotic process which were previously annotated as only “predicted,” “hypothetical” or, in some cases, were completely unannotated (9). Using flowering plants to study meiosis has some inherent methodological challenges. During reproductive development, meiosis occurs in both the male organ (anther) and the female organ (ovary). Most flowering plants bear one or more anthers in each bisexual or male flower, which make the anthers easily accessible
Wojciech P. Pawlowski et al. (eds.), Plant Meiosis: Methods and Protocols, Methods in Molecular Biology, vol. 990, DOI 10.1007/978-1-62703-333-6_20, © Springer Science+Business Media New York 2013
203
204
C. Chen and E.F. Retzel
Fig. 1 Arabidopsis anthers and male meiocytes. (a) A diagram of an anther. (b) Micrograph of an anther cross section showing the position of meiocytes (m) and surrounding layers (t tapetum; mi middle layer; en endothecium; ep epidermis). (c) A view of male meiocytes collected by the Capillary Collection Method (CCM)
and thus ideal choices to study plant meiosis. The size of anthers that undergo meiosis, however, is relatively small in plants, and this is particularly true in Arabidopsis (10). In addition, each anther contains only a small fraction of male meiocytes (pollen mother cells, Fig. 1). For instance, male meiocytes constitute about 1% of the Arabidopsis anthers, making the enrichment of meiocytes very challenging but also very desirable. Thus, several methods have been developed to investigate plant meiocyte transcriptomes. They include the following: (1) Collection of anthers that are undergoing meiosis followed by transcriptomics studies, which have been performed in several species, including Arabidopsis (11), rice (12, 13), maize (14), and wheat (15); (2) Use of genetically ablated lines (14, 16); (3) Laser capture microdissection (LCM) of meiocytes (17); and (4) Isolation of male meiocytes from meiotic anthers (9, 18). Of all these approaches, transcriptome studies using isolated meiocytes have been the most satisfying because isolation of meiocytes has significantly eliminated the background generated by non-meiotic cells of the anthers, the bias generated by abnormal development in genetically ablated lines, and the necessity of amplification of LCM samples. In addition, the isolation of staged meiocytes is much more feasible for most laboratories, as no expensive equipment such as laser dissection microscopes is required. In this chapter, we present the method that we use to profile meiotic transcriptomes of isolated male meiocytes. The procedure of meiocyte collection, which we named Capillary Collection Method (CCM), enables us to collect sufficient amounts of total RNA for transcriptome studies from highly concentrated meiocyte samples without the need for mRNA amplification. We used the RNA collected with CCM to conduct RNA-seq followed by bioinformatic analyses to identify meiosis-specific genes in Arabidopsis. We have also successfully applied this method to maize (Zea mays) transcriptome studies with slight modifications.
Meiocyte Transcriptome Analysis
2 2.1
205
Materials Microscopes
1. A stereo dissecting microscope with a 10–40× magnification. 2. An inverted microscope offering a 40–400× magnification with stage movement controls on the left side (if you are a righthanded person) or both sides, or with a motorized stage.
2.2 Capillary Collection Tubes
2.3 Collection Buffers (See Note1)
The capillary collection tubes are custom-made from disposable cotton-plugged borosilicate-glass Pasteur pipettes (Fisher Scientific, Suwanee, GA, USA) and brown rubber tubing (Fig. 2a). Each collection tube includes three parts: a glass Pasteur pipette used as a mouth piece, a connecting tube, and a capillary needle made from another glass pipette heated over a gas burner and by stretched into a capillary-like tube. 1. 1× DPBS (Dulbecco’s phosphate-buffered saline) buffer: 2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.1 mM Na2HPO4, pH 7.0 (19). 2. 0.5× Murashige and Skoog (MS) medium (20): prepared using premixed MS medium powder (Sigma Aldrich, St. Louis, MO, USA) and 20% sucrose. To use for RNA extraction, add an RNAse inhibitor to a final concentration of 1 U/μl.
2.4 Meiocyte Releasing Tools
1. Dissecting needles. 2. 1 ml disposable syringes. 3. Tweezers with flat tips.
2.5
Collection Slides
1. Fisherbrand Probeon Plus slides (Fisher Scientific, Suwanee, GA, USA). 2. Regular glass slides. 3. Fisherbrand unbreakable coverslips (Fisher Scientific, Suwanee, GA, USA).
2.6
Other Reagents
1. RNAlater (Qiagen, Hilden, Germany). 2. DNA stain (e.g., DAPI or SYTOX green). 3. Ambion RNAqueous-Micro-Kit (Life Technologies, Carlsbad, CA, USA).
2.7
Equipment
1. Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). 2. Agilent Bioanalyzer 2100 microfluidics (Agilent, Santa Clara, CA, USA).
206
C. Chen and E.F. Retzel
Fig. 2 The Capillary Collection Method (CCM). (a) A capillary collection tube. to the cotton plugs. (b) A close-up view of the meiocyte collection slide, showing how a charged slide surface can increase surface tension, which facilitates separation of meiocytes from other cells and cell debris. CNT = a collecting needle tip
3 3.1
Methods Anther Collection
1. Set a dissecting microscope on a desk at a comfortable operating height. 2. Place a Fisher Probeon slide (see Note 2) on the dissecting microscope stage, add 50 μl of collection buffer onto the slide (Fig. 2b). 3. Dissect anthers from flower buds and collect 15–20 anthers into a drop of collection buffer. Arabidopsis anthers should be at stages 5–7 (21), which correspond to stage 9 flower buds (22). Remove any anthers that do not look transparent before proceeding to the next step (see Note 3).
3.2 Capillary Collection Method
1. Set up the inverted microscope for collecting meiocytes. The microscope should have an automatically operated stage or a stage that can be easily controlled. We have modified our microscope to be equipped with a left-hand controlled stage, which is the best setting for right-handed people. 2. Release meiocytes from anthers. For Arabidopsis or other plants that have small anthers, use tweezers to break the anthers. To release the meiocytes, move the tweezers—continuously squishing—through the whole buffer drop till all anthers are
Meiocyte Transcriptome Analysis
207
broken. If anthers stick to the tweezers, use a dissecting needle to get them off back into the collection drop. Meiocytes released from anthers sink to the bottom of the buffer drop, while cell debris floats on the drop surface (Fig. 2b). 3. Transfer the slide to an inverted microscope stage. Use one hand (the right hand if you are a right-handed person) to hold the glass capillary needle and then very carefully insert the needle into the liquid drop until you touch the surface of the glass slide. Use mouth-controlled air flow through the rubber tubing to suck in meiocytes into the capillary (Fig. 2). There are two cotton plugs in the collection tube to reduce the potential of sample contamination by mouth breath (Fig. 2a). While using one hand to collect meiocytes, use the other hand to adjust the stage position to continue collecting meiocytes. In Arabidopsis, meiotic prophase meiocytes from each anther lobe stay together and form a cluster. Each cluster contains 20–50 meiocytes (Fig. 1c). 4. After collecting a number of cells and filling up the capillary, gently blow the cells into a microcentrifuge tube containing RNAlater. Multiple collections can be added into the same tube. The cells can be stored in RNAlater for 1 month at 4°C. Alternatively, pellet the cells for storage. To do this, add at least 1 volume of PBS to the microfuge tube (to dilute the dense RNAlater ® solution) and centrifuge at 400 ´ g for 1 min in a microcentrifuge. The cell pellet can be then stored at −80°C. 5. To calculate the number of collected meiocytes, gently resuspend them and pipette 1 μl of the cell suspension onto a cell counting slide. Add a DNA stain, e.g., 10 μM DAPI (Vector Laboratories, Burlingame, CA, USA) or 5 μM SYTOX green (Life Technologies, Carlsbad, CA, USA), and analyze the chromosome stage with a fluorescence microscope. 3.3 RNA Extraction from Meiocyte Samples
Extract total RNA from meiocytes using the Ambion RNAqueous Micro Kit following manufacturer’s instructions. 1. Add three volumes of 1× PBS to the meiocytes-RNAlater suspension (see Note 4). Centrifuge at 9,500 ´ g for 1 min, remove the supernatant 2. Resuspend the cell pellet by vortexing vigorously in 100 μl of lysis buffer (see Note 5). 3. Wash once with Wash Solution 1 and twice with Wash Solution 2/3. 4. Elute the RNA into a Micro-Elution tube twice with 10 μl of preheated elution buffer. 5. Measure RNA yield using the Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) and the Agilent Bioanalyzer 2100
