Extraction and labeling methods for microarrays using small amounts of plant tissue

Abstract

Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5-30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng-7 microg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies).

Comparative transcriptomics indicate changes in cell wall organization and stress response in seedlings during spaceflight

“…Tissues from shoots only were used for this study. Tissue of shoots was disrupted in a microcentrifuge tube using a Pellet Pestle motor (Kontes, Vineland, New Jersey, USA) and sterile RNase‐free pestle tip (Stimpson et al, 2009). Extraction was conducted using the RNeasy Mini Plant Kit (Qiagen, Valencia, CA) according to the procedure outlined in the RNeasy Mini Handbook.…”

“…Seedlings were shipped overnight on dry ice from −80°C storage at the University of Florida and placed in −80°C storage upon arrival. Seedlings were defrosted, then dissected in RNAlater, disrupted using a pellet pestle motor and sterile pestle, and RNA was extracted from the shoots using the RNeasy Mini Plant Kit (Qiagen, Valencia, California, USA), according to the procedure outlined previously (Stimpson et al, 2009). Following extraction, excess fluid was evaporated using a centrifugal vacuum evaporator Savant Speed Vac Concentrator (Thermo Scientific, Waltham, Massachusetts, USA).…”

An Endogenous Growth Pattern of Roots Is Revealed in Seedlings Grown in Microgravity

“…Results from the cytological and gene profiling studies will be reported in future publications; the methods used were similar to those utilized in our previously published papers (Guisinger and Kiss, 1999;Yamamoto and Kiss, 2002;Stimpson et al, 2009).…”

Exploration of plant growth and development using the European Modular Cultivation System facility on the International Space Station

“…; Stimpson et al . ). Several operation products must be developed, and integration testing is part of the experiment integration phase.…”

“…These tests often produce valuable information, worth making available to other scientists and engineers preparing for future space experiments (e.g. Correll et al 2005;Kiss et al 2007;Stimpson et al 2009). Several operation products must be developed, and integration testing is part of the experiment integration phase.…”