MasterPure™ Plant RNA Purification Kit

The MasterPure™ Plant RNA Purification Kit enables efficient purification of intact total RNA from plant tissues, including small RNAs that are typically lost when using column-based RNA purification. The protocol can be completed in less than 1 hour and may be scaled up to obtain larger amounts of RNA.

The MasterPure Plant RNA Purification Kit contains a Plant Tissue and Cell Lysis Solution designed to inactivate and remove polyphenols and polysaccharides, while releasing nucleic acids and inactivating ribonucleases. The contaminants are simply pelleted with the insoluble cellular debris, and the RNA is further purified by a simple protein precipitation step and nucleic acid precipitation step. There is no size selection with the MasterPure procedure, so small-RNA types are not lost. Unlike other plant RNA purification kits, a DNase I treatment step is included as well as reagents to remove the DNase I and remaining contaminants, so the resulting RNA is highly purified (Table 1). The purified RNA can be used directly in many molecular biology applications, e.g., end-point (Fig. 2) and real-time RT-PCR, Northern blots, miRNA analysis, and expression profiling with microarrays. Plants tested include banana, citrus, cotton, geranium, grape, maize, maple, raspberry, soybean, strawberry, and tomato leaves, alfalfa sprouts, soybean seedlings and seeds, maize seedlings and mature roots, pine needles, and field mustard seeds.

Benefits

• Optimized technology for plant tissues to remove inhibitors.
• No spin columns that reduce yields; small RNAs are purified effectively.
• Protocol is complete in less than 1 hour.
• High purity and yields of RNA.
• No phenol, chloroform, or other caustic solvents.

Figure 1. Purification of intact plant RNA from a variety of plant tissues. Plant tissue RNA was purified using the MasterPure™ Plant RNA Purification Kit, and 500 ng of each type of RNA was separated on a 1% formaldehyde gel and visualized with SYBR® Gold I. Lanes M, RNA ladder; lane 1, maize seedling RNA; lane 2, grape leaf RNA; lane 3, alfalfa sprout RNA; lane 4, strawberry leaf RNA; lane 5, raspberry leaf RNA; lane 6, pine needle RNA; lane 7, maize root RNA.

Table 1. Assessment of purity and yield of RNA purified using different methods.

Figure 2. End-point RT-PCR of RNA purified using the MasterPure™ Plant RNA Kit and other kits. RNA was reverse-transcribed using the MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit and random primers. FailSafe™ PCR was performed with PreMix D and 10 pmol of each primer, using 1 µl of each cDNA. The RT-PCR products from each type of cDNA were separated by electrophoresis and visualized with SYBR® Gold I. Lanes M, 100-bp ladder; lane 1, vendor 1; lane 2, vendor 2; lane 3, vendor 3; lane 4, MasterPure Plant RNA Kit; lane 5, MasterPure no-RT control; lane 6, no-template control.

Table 2. Yields of RNA obtained with the MasterPure™ Plant RNA Purification Kit.

Figure 3. Real-Time PCR detection of plant messages of varying abundance in RNA purified with the MasterPure™ Plant RNA Purification Kit. Maize leaf cDNA samples from Fig. 2 were used in real-time PCR with the FailSafe™ PCR System and 10 pmol of each primer. Messages detected were MatK (purple), GAPDH (green), actin 1 (red), and TubG1 (blue).