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Isolation of high-quality RNA from Platycladus orientalis and other Cupressaceae plants
Chang, Ermei; Zhao, Yuxin; Wei, Qianchun; Shi, Shengqing & Jiang, Zeping
Abstract
Platycladus orientalis has a lifespan of several thousand years in China, making it a good plant in which to study
aging at the molecular level, but this requires sufficient quantities of high-quality P. orientalis RNA. However, no
appropriate methods have been reported for total RNA isolation from P. orientalis leaves. The TRIzol method did
not extract RNA, while cetyltrimethylammoniumbromide, sodiumdodecyl sulfate-phenol, and plant RNAout kit
(Tianz, Inc., China) protocols resulted in low yields of poor quality RNA. Isolating total RNA using the Spectrum™
Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) resulted in a high-quality product but a low yield.
However, the two-step removal of polyphenols and polysaccharides in the improved plant RNAout kit protocol
resulted in the isolation of RNA with a 28S:18S rRNA ratio of band intensities that was ~2:1, the A260/A280
absorbance ratio was 2.03, and the total RNA yield from P. orientalis leaves was high. This protocol was tested
on different P. orientalis tissues of different ages and on leaves of five other Cupressaceae plants. The total
RNAs were successfully used in complementary DNA synthesis for transcriptome sequencing and would be
suitable to use in additional experiments. The results of this study will benefit future studies in Cupressaceae
plants.
Keywords
Cupressaceae plants; Polysaccharides; Polyphenols; Total RNA isolation; Two-step removal of polyphenols and polysaccharides
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