The overall goal of this procedure is to extract high purity RNA from plant seeds. This method can help answer key questions in the cereal biology field, such as understanding gene profiles, containing the original transcripts in seeds. The main advantage of this technique is that researchers are able to prepare high-purified RNA with slight modification to the spin column-based method.
Generally, individuals new to this method will struggle because the high amount of oils, proteins, carbohydrates and polyphenols in front sieve make it difficult to isolate highly purified RNA;demonstrating the procedure will be Kazumi Hikino, a technician from our laboratory. To begin the experiment, add one percent weight per volume molecular biology grade PVP to cell lysis buffer for RNA extraction, and vortex the mixture vigorously. Incubate the PVP for 20 minutes at 25 degrees celsius to dissolve it completely.
After 20 minutes of incubation, mix the buffer gently by turning the tube upside down to prevent bubbles from forming. Harvest fruits from Arabidopsis thaliana plants and place them into two 1.5 millimeter polypropylene tubes on ice, then place the fruits on aluminum plates kept at four degrees celsius and isolate seeds from the fruits under a stereo microscope. Next, place approximately 200 isolated seeds into 1.5 milliliter polypropylene tubes that have been previously stored in an aluminum rack on ice, and immediately place the tubes in liquid nitrogen.
Remove the tubes from the liquid nitrogen and return them to the aluminum rack on ice. Add 100 microliters of the one percent PVP buffer to each tube and centrifuge the tubes for one minute at 1000 times G, at four degrees celsius. After centrifugation, homogenize the sample with a stainless steel pestle using a motor grinder for 60 seconds while keeping the tube in the aluminum rack on ice;next, add 550 microliters of the 100%PVP buffer and gently turn the tube upside down to mix and incubate the tube.
After incubation, centrifuge the solution then transfer 550 microliters of the supernatant to a new 1.5 millimeter polypropylene tube and centrifuge the supernatant. Transfer 450 microliters of the supernatant to a new 1.5 millimeter polypropylene tube. Use the supernatant as the cell lysate for RNA extraction using a commercially available kit.
Prepare an aluminum rack on ice, and thaw the total RNA mix by gently tapping, then place the tube on the rack. Measure the RNA concentration using a microvolume spectrophotometer. Then dilute the total RNA in RNase-free water.
Thaw the total RNA and buffer sets from the commercial kit, keep the enzymes from the kit on ice after gentle tapping. Prepare sterilized nuclease free 0.2 milliliter polypropylene tubes for subsequent analysis. On ice, combine five microliters of total RNA, one microliter or 50 micromolar of oligo DT primer and one microliter, or 50 micromolar random six-mer in the 0.2 milliliter polypropylene tube.
Incubate the mixture for 15 minutes at 37 degrees celsius, then place the tube on ice. Adjust the plasmid concentrations for DNA templates for the standard curves. Next, dilute the CDNA solutions at one to 100 with distilled water, then add two microliters of the diluted CDNA solutions and the previously prepared plasmids for the standard curves to the master mix from the quantitative realtime PCR kit.
Finally, set up the realtime PCR and insert the samples. Total RNA was isolated from approximately 1, 000 seeds using varying concentrations of cell lysis buffer, PVP. The quantities and purity of the isolated RNAs demonstrate that 1.0%PVP is the most effective concentration to isolate large amounts of purified RNA from seeds.
The concentrations, A260 and A280 ratios, and A260 A230 ratios of RNA isolated from 200 seeds showed that this method was best at isolating highly-purified RNA from eight and 12 days after flowering, or DAF seeds. Although the amounts of wrinkled one and sugar-dependent one transcripts are relatively low in developing seeds, it was possible to detect expression changes between seeds at different developmental stages via realtime PCR;this method is also suitable for other oil seeds, as demonstrated in this table. After its development, this technique paved the way for researchers in the field of plant biology to explore gene expression profiles in high concentration of stored reserves, such as seeds and fruits.
