Guidelines for RNA Purification from Plants
- Fresh or frozen plant tissue can be processed with the Monarch Total RNA Miniprep Kit (NEB T#2010).
- Use an appropriate amount of starting material.The maximum input for plant tissue is 100 mg. Using too much sample may reduce lysis efficiency, introduce excessive amounts of cellular components other than RNA, and compromise RNA binding to the RNA Purification Column.
- Frozen and fresh tissue can be pulverized into a powder using a mortar and pestle. The mortar and pestle are pre-chilled on dry ice and liquid nitrogen is poured into the mortar to snap freeze the sample while pulverizing. Keep pulverized powders frozen until mixed with Monarch DNA/RNA Protection Reagent (NEB #T2011). Samples can then be lysed/homogenized by bead homogenization.
- Keep frozen samples frozen until mixed with DNA/RNA Protection Reagent. After addition of Protection Reagent, it is important to proceed immediately to mechanical lysis (e.g., bead homogenization). If necessary, samples in Protection Reagent can be placed on ice briefly until they can be processed.
- Be sure to use a 1X solution of Monarch DNA/RNA Protection Reagent. The supplied 2X concentrate can be diluted with nuclease-free water.
- Proteinase K digestion is not required for processing plant samples.
- Addition of Monarch RNA Lysis Buffer (NEB #T2012) and all subsequent steps should be performed at room temperature.
- When processing plant samples, a larger volume of DNA/RNA Protection Reagent is required. As a result, larger volumes of RNA Lysis Buffer and ethanol are also required, and columns must be reloaded. To reduce sample volumes and reloading of columns, RNA Lysis Buffer can be used for mechanical lysis instead of DNA/RNA Protection Reagent