A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection

Abstract

Laser microdissection (LM) combined with microarray analysis or next-generation sequencing of cDNA is a powerful tool for understanding molecular events in individual cell types of plants as well as animals. Obtaining high quality RNA is essential for this approach. For plant tissues, paraffin-embedded sections better preserve cell structure than do frozen sections. However, the conventional method for preparing paraffin sections is a lengthy process involving embedding the tissue and floating and drying the sections, during which time RNA degradation occurs. Here, we describe a method for preparing serial sections that greatly reduces RNA degradation: we reduced (1) the embedding time from 4-6 days to about 5 h by using a recently developed microwave method; (2) the time of floating sections from ~10 min to less than 5 min, (3) the drying time from ~12 to 1 h; and (4) the drying temperature from 42 to 4°C. With this method, we were able to isolate higher integrity RNA from many kinds of plant tissues than is typically obtained by the conventional paraffin preparation method. The improvement in RNA quality and yield removes a major obstacle to the widespread use of LM with high-throughput technologies for plants.

Inflorescence Meristem Identity in Rice Is Specified by Overlapping Functions of Three AP1/FUL-Like MADS Box Genes and PAP2, a SEPALLATA MADS Box Gene

“…Shoot apices were collected at different growth stages. The samples were fixed in 75% ethanol and 25% acetate and embedded in paraffin using a microwave processor (Energy Beam Sciences) according to Takahashi et al (2010). The paraffin-embedded samples were cut into 10-µm-thick sections using a microtome (Leica RM2255).…”

RNA Sequencing of Laser-Capture Microdissected Compartments of the Maize Kernel Identifies Regulatory Modules Associated with Endosperm Cell Differentiation

“…Fixed kernels were then dehydrated in a graded ethanol series, cleared in n-butanol, embedded in Paraplast X-tra (McCormick Scientific Leica) using microwave (Takahashi et al, 2010), cut to 10-mm sections, and mounted on PEN-coated slides. Shortly before capture, sections were deparaffinized in Xylenes and air-dried (Takahashi et al, 2010).…”

“…Fixed kernels were then dehydrated in a graded ethanol series, cleared in n-butanol, embedded in Paraplast X-tra (McCormick Scientific Leica) using microwave (Takahashi et al, 2010), cut to 10-mm sections, and mounted on PEN-coated slides. Shortly before capture, sections were deparaffinized in Xylenes and air-dried (Takahashi et al, 2010). Individual cell types or kernel compartments were captured directly into an aliquot of Arcturus PicoPure RNA extraction buffer (Applied Biosystems) using a Leica LMD6500 laser microdissection system (Leica Microsystems).…”

SEMI-ROLLED LEAF1 Encodes a Putative Glycosylphosphatidylinositol-Anchored Protein and Modulates Rice Leaf Rolling by Regulating the Formation of Bulliform Cells

“…Leaves of wild-type and srl1-1 seedlings at 5 DAG were dissected, fixed by a fixation solution (ethanol:acetate, 75:25), and embedded by Paraplast plus (Sigma-Aldrich) as described (Takahashi et al, 2010). Cross sections (10 mm) were cut on a rotary microtome (Leica Microtome), floated on the RNAsecure (Ambion) solution on the polyethylene naphthalate membrane glass slides (Leica).…”