A Method for Extracting High-Quality RNA from Diverse Plants for Next-Generation Sequencing and Gene Expression Analyses

• Main Authors: Roxana Yockteng, Ana M. R. Almeida, Stephen Yee, Thiago Andre, Colin Hill, Chelsea D. Specht
• Format: Article
• Language: English
• Published: Wiley 2013-12-01
• Series: Applications in Plant Sciences
• Subjects: cDNA library; gene expression; Illumina; RNA-Seq; RNA extraction; transcriptome
• Online Access: http://www.bioone.org/doi/full/10.3732/apps.1300070

Premise of the study: To study gene expression in plants, high-quality RNA must be extracted in quantities sufficient for subsequent cDNA library construction. Field-based collections are often limited in quantity and quality of tissue and are typically preserved in RNA<i>later</i>. Obtaining sufficient and high-quality yield from variously preserved samples is essential to studies of comparative biology. We present a protocol for the extraction of high-quality RNA from even the most recalcitrant plant tissues. Methods and Results: Tissues from mosses, cycads, and angiosperm floral organs and leaves were preserved in RNA<i>later</i> or frozen fresh at −80&#176;C. Extractions were performed and quality was measured for yield and purity. Conclusions: This protocol results in the extraction of high-quality RNA from a variety of plant tissues representing vascular and nonvascular plants. RNA was used for cDNA synthesis to generate libraries for next-generation sequencing and for expression studies using quantitative PCR (qPCR) and semiquantitative reverse transcription PCR (RT-PCR).