220.127.116.11.1. Nosema DNA extraction
DNA extraction can be performed from specific tissue (e.g. ventriculus, rectum, fat body), subdivided bee sections (e.g. metasoma), whole bees, or homogenates from pooled samples (see the BEEBOOK paper on bee anatomy and dissection (Carreck et al., 2013)).
Nosema DNA extraction from bee homogenates:
- Crush fresh or flash frozen tissue to generate a homogeneous homogenate of
bee/bee guts. For example,
1.1. place a maximum of 30 bees in a filter grinding bag (e.g. extraction bags from BIOREBA AG, Switzerland).
1.2. Add 0.5 ml (DNAase/RNAase free) ddH2O per bee
1.3. homogenize the mixture using a homogenizer (e.g. Homex6 from BIOREBA AG; Switzerland).
1.4. Flash-freeze in liquid nitrogen is possible prior to homogenization to aid in mechanically breaking open cells.
1.5. Crush the sample
Without access to a robot, one can use a pestle to crush the bee tissue.
- Transfer 100 µl of the liquid homogenate into a microcentrifuge tube and centrifuge for 3 min at 16,100 g to precipitate the microsporidia and other cellular material.
- Discard the supernatant.
- Freeze the pellet by using liquid nitrogen
- Crush using a pestle until pulverized (in order to break open Nosema spore walls),
- Repeat steps 4 and 5 two or three times so that Nosema DNA goes into solution.
- Use the DNeasy® Plant Mini kit
protocol (Qiagen) following the Mini protocol for plant tissue to extract DNA
from the homogenate.
Other non-proprietary DNA extraction protocols (e.g. those using phenol/chloroform; chelex resin; Tay et al. 2005) were used with poor success, possibly because bee guts contain plant secondary compounds and tissue may be in a state of decay due to poor preservation. Research on other extraction techniques (e.g. using CTAB) is needed to provide cheaper yet efficient methods for the extraction of Nosema DNA from bees.
- Complete the final elution step in 100 µl of 0.01M Tris (pH 7.5) buffer.
The same Qiagen protocol can also be implemented in the QiaCube (Qiagen) for automated DNA extraction.