Journal
APPLICATIONS IN PLANT SCIENCES
Volume 1, Issue 12, Pages -Publisher
BOTANICAL SOC AMER INC
DOI: 10.3732/apps.1300070
Keywords
cDNA library; gene expression; Illumina; RNA-Seq; RNA extraction; transcriptome
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Funding
- Evolutionary Genetics Lab, Museum of Vertebrate Zoology
- National Science Foundation [IOS 0845641, DEB 1110461]
- University of California Institute for Mexico (UC MEXUS)-Consejo Nacional de Ciencia y Tecnologia (CONACYT)
- Hellman Family Fund
- Prytanean Junior Faculty Award
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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 later. 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 later or frozen fresh at -80 degrees 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).
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