RNA extraction from plant tissues presents many more challenges than from animal cells or tissues. This is because plant tissues are quite variable and represent a large number of different species. Plant tissue cell walls are often made up of various compounds and arrangements (celluloses, hemicelluloses, pectin’s), therefore finding one protocol that works well with all of these variations is challenging. Furthermore, plant tissues contain various substances that interfere with RNA extractions and/or cause interferences or inhibition in downstream application. Compounds such as phenolics, phytotoxins, polysaccharides, starches, tannins, and other secondary metabolites are present in various amounts and combinations, making it difficult for most RNA and even DNA extraction protocols to yield high quality nucleic acid for downstream applications. Therefore, many plant RNA purification companies have adapted a dual lysis buffer system, which contains two lysis buffers that both have a unique range of plant materials that they are able to work with. The issue with this system is that a process of trialand- error must be carried out to see which buffer works most effectively on a sample, at the customers cost.
Norgen’s Plant/Fungi RNA Purification Kit contains a universal, robust lysis buffer that is very effective over a wide range of plant species. It allows Norgen’s Plant/Fungi RNA Purification to target the isolation of RNA from various challenging plant samples, including grape and pine needle, with no need to test different lysis buffers. This saves the customer’s time and effort to establish a rapid RNA purification system in the lab.
With Norgen’s universal lysis buffer, plant total RNA can be purified from a wide range of fresh or frozen plant tissues, plant cells, or filamentous fungi samples using this kit. Furthermore, the total RNA profile of the sample is purified, from large mRNA and ribosomal RNA, down to microRNA (miRNA) and small interfering RNA (siRNA) using Norgen’s proprietary resin as the separation matrix. The procedure is also free of environmentally hazardous compounds such as phenols and chloroform, and yet maintains the integrity of its total RNA profile isolation. The yield and quality of the isolated RNA is of the highest integrity, and is excellent for use in downstream applications such as RNA sequencing, Next Generation sequencing, RT-PCR, qRT-PCR, icroarrays, Northern blots for host RNA (miRNA) or for pathogen detection, among many other applications.
In this application note, we show the effectiveness of Norgen’s universal lysis buffer to successfully isolate high quality RNA from various plant species, including challenging samples, and we compare it to a popular commercially available kit from Qiagen. Many publications showing the utility of Norgen’s universal buffer and kit for various plant species and applications are listed on our website: (www.accuvisbio.com )
MATERIALS AND METHODS
Plant RNA Isolation
Plant RNA was isolated from 50 mg of plant leaf tissue (equivalent to ~5 x 106 plant cells) using Norgen's Plant/Fungi RNA Purification Kit as per the provided protocol (Figure 1). Briefly, the plant leaf tissue (apple,peach, grape, pine needle, strawberry and pear) was ground in a mortar, containing enough liquid nitrogen to cover the sample, with a pestle until the tissue was ground into a fine powder. Next, the universal lysis buffer was added. The lysate was then transferred into a filter column, assembled with an RNAse-free microcentrifuge tube and centrifuged for 1 minute at 14,000 x g (~14,000 rpm) to filter out cellular debris. The flow-through was then transferred to a new RNAse-free microcentrifuge tube and an equal volume of 96-100% ethanol was added and mixed by vortexing. Next, 600 μL of the clarified lysate was then loaded onto an assembled column and centrifuged for 1 minute at 14,000 x g (~14,000 rpm). The flow-through was discarded and the column was reassembled. The remaining lysate was then loaded onto the column and re-centrifuged for 1 minute at 14,000 x g. The column was then washed a total of three times by applying 400 μL of Wash Solution to the column, centrifuging for 1 minute at 14,000 x g (~14,000 rpm) and then discarding the flow-through. Columns were centrifuged for 2 minutes at 14,000 x g (~14,000 rpm) to thoroughly dry the resin. For RNA elution the column was placed into a fresh 1.7 mL elution tube and 50 μL of the elution buffer was applied to the column. Columns were then centrifuged for 2 minutes at 200 x g (~2000 rpm), followed by a 2 minute spin at 14,000 x g. Purified RNA was then stored at -20°C for several days or at -70°C for long term storage.
At the same time, RNA was purified from plant leaf tissue using the leading market competitor's plant RNA purification kit according to the manufacturer's protocol and used in comparative experiments.