Difference between revisions of "IC4R014-RNA-Seq-2013-23322175"

Revision as of 13:51, 13 July 2016

Transcriptomic analysis of rice (Oryza sativa) endosperm using the RNA-Seq technique

The endosperm plays an important role in seed formation and germination, especially in rice (Oryza sativa). In this project, the researchers used a high-throughput sequencing technique (RNA-Seq) to reveal the molecular mechanisms involved in rice endosperm development.

The endosperm were derived from rice (O. sativa L.ssp. indica cv.9311), which were grown in a greenhouse at Wuhan University, China. Caryopses were tagged at the initiation of pollination. The samples consisted of three replicates arranged in a randomized complete block design. For each replicate, development seed were collected from 15 different plants at 3, 6, and 10 DAP during the growing season. The three-stage endosperm was taken from caryopses with microdissection needles under a dissection microscope (Olympus, Tokyo, Japan). The materials were immediately frozen in liquid nitrogen and kept at -80℃ until required (for RNA extractions).

Total RNA was prepared using Trizol according to the manufacturer’s protocol (Invitrogen, Burlington, ON, Canada). The yield and purity of each RNA sample was determined by the absorbance (Abs) at 260 and 280 nm and the integrity of all RNA samples was monitored on 1 % agarose gels. The total RNA samples were pooled into three-stage samples and 10 lg of total RNA from each sample was used to isolate poly (A) mRNA and to prepare a nondirectional Illumina RNA-Seq library with an mRNASeq 8 Sample Prep Kit (Illumina). Each library had an insert size of 200 bp, and 42–50 bp sequences were generated via Illumina HiSeqTM 2000.

State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China

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 ==Illumina Sequencing==
+* Total RNA was prepared using Trizol according to the manufacturer’s protocol (Invitrogen, Burlington, ON, Canada). The yield and purity of each RNA sample was determined by the absorbance (Abs) at 260 and 280 nm and the integrity of all RNA samples was monitored on 1 % agarose gels. The total RNA samples were pooled into three-stage samples and 10 lg of total RNA from each sample was used to isolate poly (A) mRNA and to prepare a nondirectional Illumina RNA-Seq library with an mRNASeq 8 Sample Prep Kit (Illumina). Each library had an insert size of 200 bp, and 42–50 bp sequences were generated via Illumina HiSeqTM 2000.
 ==Research Findings==