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Simple and Rapid Isolation of Plant RNA,,, Using the Eppendorf Perfect RNA, Eukaryotic, Maxi Kit

uid nitrogen for a minimum of 10 min. and then transferred into a fresh mortar (to prevent the buffer from freezing). All further procedural steps were performed according to the protocol of the Perfect RNA Maxi Kit, with a slight modification to Steps 13 (see protocol below). Quantity and quality of the obtained RNA were determined based on their UV absorptions at A260 nm and A280 nm. Two g of each RNA sample eluate were analyzed by agarose gel electrophoresis. RNA samples were concentrated by ethanol precipitation for subsequent RT-PCR amplification of two Arabidopsis-specific gene fragments. Two g of total RNA from an Arabidopsis leaf/flower/pod mixture were used in a 20 l RT reaction. Two l of the RT reactions were used as a template for the 50 l PCR (Two-step RT-PCR). cDNA synthesis was performed with a Thermoscript RT-PCR-System (Invitrogen/Life Technologies) according to the manufacturer's protocol. PCR was performed with Ex Taq (TaKaRa) under the following conditions:

Initial incubation at 96C for 4 min.; 36 cycles:

  1. Denaturation at 94C, 30 sec.
  2. Annealing at 64C, 30 sec.
  3. Elongation at 72C, 2.5 min.
  4. Final incubation at 72C for 5 min.
Protocol
  1. Lyse cells by adding 9 ml lysis solution to the mortar and homogenize for approximately 1 min. using a pestle.
  2. Transfer lysate into a 50 ml centrifuge tube which can withstand 3,000 x g. Add an additional 9 ml of lysis solution and vortex vigorously.
  3. Incubate homogenized cells or tissue for at least 5 min. at room temperature. Centrifuge at 3,000 x g for 20 min. Transfer supernata
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