An optimized protocol for isolation of high‐quality RNA through laser capture microdissection of leaf material

Abstract

Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA . Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low‐melting‐point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high‐quality RNA suitable for down‐stream applications such as quantitative reverse transcriptase–polymerase chain reactions or RNA ‐sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.

The bundle sheath of rice is conditioned to play an active role in water transport as well as sulfur assimilation and jasmonic acid synthesis

“…Sections (1 cm) from the middle of the fourth fully expanded leaves were sampled 4 h after dawn. Leaf tissue was fixed and embedded into Steedman’s wax as described previously (Hua and Hibberd, 2019), with minor modifications. For example, rice leaves were fixed in 100% (v/v) acetone on ice for 4 h and before embedding tissue infiltrated with 100% Steedman’s wax at 37°C overnight.…”

“…Here we used an optimized LCM protocol for RNA isolation from leaves (Hua and Hibberd, 2019) to study the patterns of gene expression in bundle sheath, veinal (including phloem and xylem parenchyma, xylem, as well as sieve elements and companion cells) and mesophyll cells of rice. We had three main hypotheses.…”

Photosynthesis-independent production of reactive oxygen species in the rice bundle sheath during high light is mediated by NADPH oxidase

“…For LCM, leaf samples were cut into ∼5-mm pieces and fixed in 100% (vol/vol) ice-cold acetone at 4°C overnight. The next day, samples were dehydrated and embedded with Steedman's polyester wax (68). Blocks were sectioned using a rotary microtome to 8-μm thickness and then floated on Arcturus polyethylene naphthalate (PEN) membrane slides (Thermo Fisher Scientific) with diethylpyrocarbonate (DEPC)-treated water.…”

The bundle sheath of rice is conditioned to play an active role in water transport as well as sulfur assimilation and jasmonic acid synthesis

“…For RNA in situ hybridization, middle sections from the fourth fully expanded leaf were sampled and fixed overnight using FAA fixative (50% (v/v) ethanol, 5% (v/v) acetic acid and 3.7% (v/v) formaldehyde. They were then dehydrated through an ethanol series of 50%, 70%, 85%, 95% and 100% (v/v) and embedded into Steedman's wax as described previously (Hua and Hibberd, 2019). 8 micron thick sections were obtained on a microtome and mounted onto Superfrost plus slides.…”

“…Here we used an optimised laser capture microdissection protocol for RNA isolation from leaves (Hua and Hibberd 2019) to study the patterns of gene expression in bundle sheath, veinal (including phloem and xylem parenchyna, xylem, as well as sieve elements and companion cells), and mesophyll cells of rice. We had three main hypotheses.…”