Name: experimental design for laser microdissection rna-seq: lessons from an analysis of maize leaf development
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The authors thank S. Hake for ongoing collaboration and stimulating discussions about ligule development. This work is supported by National Science Foundation Grants MCB 1052051 and IOS-1848478.

NOTE: Fix tissue for histological analysis at the same time that tissue is fixed for LM. Examine stained sections for morphological features that will guide later LM. When comparing mutant to wild-type, perform in situ hybridization or immunolocalization to define the domain where the gene of interest is expressed (in this case lg1).
1. Tissue Fixation and Processing
<ol>
	<li>Grow flats of maize seedlings to two weeks old under standard conditions6....

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+modules+controlling+maize+inflorescence+architecture.">Regulatory modules controlling maize inflorescence architecture.</a> Genome Res. 24 (3), 431-443 (2014).</li><li>Takacs, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+maize+shoot+apical+meristem.">Ontogeny of the maize shoot apical meristem.</a> Plant Cell. 24 (8), 3219-3234 (2012).</li><li>Aida, M., Ishida, T., Tasaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoot+apical+meristem+and+cotyledon+formation+during+Arabidopsis+embryogenesis:+interaction+among+the+CUP-SHAPED+COTYLEDON+and+SHOOT+MERISTEMLESS+genes.">Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes.</a> Development. 126 (8), 1563-1570 (1999).</li><li>Ishida, T., Aida, M., Takada, S., Tasaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+CUP-SHAPED+COTYLEDON+genes+in+gynoecium+and+ovule+development+in+Arabidopsis+thaliana.">Involvement of CUP-SHAPED COTYLEDON genes in gynoecium and ovule development in Arabidopsis thaliana.</a> Plant Cell Physiol. 41 (1), 60-67 (2000).</li><li>Takada, S., Hibara, K., Ishida, T., Tasaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CUP-SHAPED+COTYLEDON1+gene+of+Arabidopsis+regulates+shoot+apical+meristem+formation.">The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation.</a> Development. 128 (7), 1127-1135 (2001).</li><li>Nardmann, J., Ji, J., Werr, W., Scanlon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maize+duplicate+genes+narrow+sheath1+and+narrow+sheath2+encode+a+conserved+homeobox+gene+function+in+a+lateral+domain+of+shoot+apical+meristems.">The maize duplicate genes narrow sheath1 and narrow sheath2 encode a conserved homeobox gene function in a lateral domain of shoot apical meristems.</a> Development. 131 (12), 2827-2839 (2004).</li><li>Bonner, W. A., Hulett, H. R., Sweet, R. G., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+activated+cell+sorting.">Fluorescence activated cell sorting.</a> Rev Sci Instrum. 43 (3), 404-409 (1972).</li><li>Birnbaum, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+map+of+the+Arabidopsis+root.">A gene expression map of the Arabidopsis root.</a> Science. 302 (5652), 1956-1960 (2003).</li><li>Brady, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+root+spatiotemporal+map+reveals+dominant+expression+patterns.">A high-resolution root spatiotemporal map reveals dominant expression patterns.</a> Science. 318 (5851), 801-806 (2007).</li><li>Carter, A. D., Bonyadi, R., Gifford, M. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant microtechnique and microscopy. , (1999).</li><li>Jackson, D., Veit, B., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+maize+KNOTTED1+related+homeobox+genes+in+the+shoot+apical+meristem+predicts+patterns+of+morphogenesis+in+the+vegetative+shoot.">Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot.</a> Development. 120, 405-413 (1994).</li><li>Javelle, M., Marco, C. 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Experimental design is a critical factor in RNA-seq experiments. Key considerations are the precise domain(s) and developmental stage(s) to be analyzed, and what comparisons will be made. It is crucial to think in terms of comparisons, since the output is typically a list of genes that are DE between two or more conditions. As with all experiments, it is important to alter only one variable at a time. For example, when comparing different leaf domains, leaves of the same age and developmental stage, grown under the same ...

The maize leaf is an ideal model to study the formation of developmental fields during morphogenesis, as it has a distinct boundary between the blade and sheath that is amenable to genetic dissection (Figure 1A). During the early stages of leaf development, a linear band of smaller cells, the preligule band (PLB), subdivides the leaf primordium into pre-blade and pre-sheath domains. A fringe-like ligule and triangular auricles develop from the PLB (Figure 1A, C, D). Genetic screens have identified mutations that disrupt the blade-sheath boundary. For example, recessive liguleless1 (lg1) mutations delete the ligule and auricles1,2,3,4 (Figure 1B). In situ hybridization revealed that lg1 transcript accumulates at the PLB and emerging ligule, making it an excellent marker for ligule development5,6 (Figure 1E).
<img alt="Figure 1" src="/files/ftp_upload/55004/55004fig1.jpg" /> 
Figure 1: Wild-type and liguleless1-R maize leaves. (A) Blade-sheath boundary region of mature wild-type leaf showing ligule and auricle structures. (B) Blade-sheath boundary region of mature liguleless1-R leaf showing absence of ligule and auricle structures. Leaves in A and B have been cut in half along the midrib. (C) Longitudinal section through wild-type leaf primordium. Sample has been processed and stained for histological analysis. The initiating ligule is apparent as a bump protruding from the plane of the leaf (arrowhead). (D) Longitudinal section through wild-type leaf primordium. Sample has been processed for LM as described in the text. Arrowhead indicates initiating ligule. (E) lg1 in situ hybridization of shoot apex lateral longitudinal section. Asterisks indicate lg1 transcript accumulation at the PLB of the P6 leaf primordium. Arrows indicate base of P6 primordium. Bar indicates measurement from the base of the primordium to the PLB. Scale bars in A and B = 20 mm. Scale bars in C-E = 100 &#181;m. This figure has been modified from reference6(Copyright American Society of Plant Biologists). <a href="http://cloudflare.jove.com/files/ftp_upload/55004/55004fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this study, LM RNA-Seq was employed to identify a suite of genes that are differentially expressed (DE) at the blade-sheath boundary relative to other parts of the leaf primordium and to identify genes that are DE in lg1-R mutants relative to wild-type siblings. LM RNA-Seq is a method of quantifying transcript accumulation in specific cells or cellular domains7. LM systems combine a laser and a microscope with a digital camera. Sectioned tissue is mounted on slides and viewed through the microscope. The LM software typically includes drawing tools that allow the user to outline any selected region for microdissection. The laser cuts along the line, and the selected tissue is catapulted off the slide and into a tube suspended above the slide. LM allows the user to microdissect precise domains, including specific cell layers and even single cells8,9. RNA can then be extracted from the microdissected tissue. Subsequently, the RNA-Seq component utilizes next-generation sequencing to sequence cDNA libraries generated from the extracted RNA10,11.
Key advantages of LM RNA-seq are the ability to quantify transcript accumulation in precisely defined domains and the capacity to profile the entire transcriptome simultaneously7. The technique is particularly suited to probing early developmental events where the region of interest is often microscopic. Previous studies have utilized LM combined with microarray technology to study developmental processes in plants9,12,13. RNA-Seq has the advantage of quantifying transcripts across a broad dynamic range, including low-expressed genes, and prior sequence information is not required10,11. Moreover, LM RNA-Seq has the potential to highlight developmentally important genes that may be missed in mutagenesis screens due to genetic redundancy or to lethality of the loss-of-function mutant.
Developmentally important genes, such as narrow sheath1 (ns1) and cup-shaped cotyledon2 (cuc2), often have specific expression patterns of just one or a few cells17,18,19,20. Many are expressed only during early developmental stages and not in the mature organ. When whole organs or large domains are analyzed, these cell-specific transcripts are diluted and may not be detected in more conventional analyses. By permitting analyses of precisely defined domains, LM RNA-Seq enables these tissue-specific genes to be identified and quantified.
Crucial factors in the success of the experiments described here were a thorough histological analysis that guided selection of the appropriate developmental stage and domain for analysis, and precise measurement of cell-tissue domains for LM. To ensure that equivalent domains were sampled for all replicates, tissue was collected from leaf primordia at the same developmental stage and the microdissected domains were measured relative to morphological landmarks such as the emerging ligule (Figure 2). It is known that some genes are expressed in a gradient from the tip to the base of the leaf. By measuring precise domains, variation due to sampling from different locations along the leaf proximal-distal axis was kept to a minimum (Figure 3A). By microdissecting domains of the same size, variation due to differential dilution of cell-specific transcripts was also reduced (Figure 3B). Lateral longitudinal sections of the shoot apex were used for all microdissections. These are sections that are perpendicular to the midrib-margin axis (Figure 4). Using only sections that include the SAM ensures that equivalent lateral regions of leaf primordia are analyzed.
In samples processed and sectioned for LM, the first morphological sign of ligule outgrowth is a bump on the adaxial side due to periclinal cell divisions in the adaxial epidermis (Figure 1D, Figure 2). It was determined that the emerging ligule could be reliably identified at plastochron 7 stage leaf primordia. We were interested in genes expressed in the entire ligule region, including the emerging ligule and the cells immediately distal that will form the auricle. In order to ensure that equivalent tissue selections were made, the ligule bump was used as a morphological landmark and a 100 &#181;m rectangle centered on the ligule bump was selected for LM (Figure 2A, 2B). Equivalent sized rectangles of pre-blade and pre-sheath were selected from the same leaf primordia.
Analyses of liguleless mutant plants presented a different challenge; lg1-R mutants do not form a ligule, therefore this morphological feature could not be used to select the region for LM. Instead, the domain of lg1 transcript accumulation in wild-type leaf primordia was determined, and a region that would encompass this domain was defined. These preliminary analyses were performed on seedlings from the same planting as were used for the final analysis, since previous work has shown that the location of the PLB varies depending on growth conditions. In situ hybridization indicated that lg1 transcripts accumulate in the PLB of P6 leaf primordia (Figure 1E). We selected a domain 400-900 &#181;m from the base of the leaf primordia that encompassed the domain of lg1 expression (purple rectangles, Figure 2A) and captured these equivalent regions from wild-type and lg1-R plants. To minimize variation in genetic background and growth conditions when comparing transcript accumulation in lg1-R and wild-type plants, segregating families of mutants and wild-type siblings were used.

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			<td height="63" style="height:63px;width:247px;">PicoPure RNA Isolation Kit</td>
			<td style="width:81px;">ThermoFisher Scientific</td>
			<td style="width:136px;">KIT0204</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental design for laser microdissection rna-seq: lessons from an analysis of maize leaf development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Using the LM scheme outlined in Figure 2, approximately 1,000,000-1,500,000 &#181;m2 of tissue was collected for each replicate in the all-cell-layers LM (Figure 5), and 200,000 &#181;m2 per replicate for the adaxial epidermis LM. Approximately 2,500,000 &#181;m2 of tissue was collected for each replicate in the LM of lg1-R and wild-type leaf primordia. Two rounds of linear RNA amplification yielded microgram quan...

Watch this Scientific Journal Video about Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development at JoVE.com

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

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Biochemistry

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