Guidelines for RNA Purification from Bacteria
- Fresh or frozen bacteria can be processed with the Monarch Total RNA Miniprep Kit (NEB #T2010).
- Use an appropriate amount of starting material. The maximum input for bacteria is 1x109 cells. Using too much sample can reduce lysis efficiency, introduce excessive amounts of cellular components other than RNA, and compromise RNA binding to the RNA Purification Column. Cells grown in liquid culture should be pelleted and all culture medium removed prior to sample disruption and homogenization.
- Mechanical lysis using a bead homogenizer is recommended for gram-positive bacteria, and some gram-negative strains. Alternatively, bacteria may be lysed enzymatically with a glycanase (e.g., lysozyme). Protocols for mechanical or enzymatic lysis can be found in the product manual.
- Some gram-negative bacteria (e.g., E. coli) do not require mechanical lysis and can be lysed directly in 1X Monarch DNA/RNA Protection Reagent
- Be sure to use a 1X solution of Monarch DNA/RNA Protection Reagent. The supplied 2X concentrate can be diluted with nuclease-free water.
- 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, place samples in Protection Reagent on ice briefly until they can be processed.
- Addition of RNA Lysis Buffer and all subsequent steps should be performed at room temperature.
- Proteinase K digestion is not required for processing bacteria samples.
- When processing higher sample input amounts (i.e., 5 x107 – 1 x109 cells), a larger volume of 1X 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.