8.3.1. Protocol 1 – affinity column purification

The processing consists of making a primary homogenate from 1-30 bees and purifying RNA from 100 μl aliquots of the homogenate (equivalent to 20 mg bee tissue: the maximum loading capacity of one affinity column). Based on the Qiagen column procedures. β-mercaptoethanol is toxic. 

1. Prepare fresh GITC buffer:  
1.1.  5,25 M guanidinium thiocyanate (guanidine isothiocyanate),
1.2.  50 mM TRIS.Cl (pH 6.4),
1.3.  20 mM EDTA,
1.4.  1,3% Triton X-100,
1.5.  1% β-mercaptoethanol.
2. Place frozen bees in the homogenizer of choice.  
3. Per bee add the following amount of GITC buffer:




Total volume   

Worker bee

120 mg

500 μl

600 μl


180 mg

700 μl

900 μl

Worker pupa  

160 mg

650 μl

800 μl

Drone pupa

240 mg

1000 μl  

1200 μl

5. Proceed according to the Plant RNA extraction protocol using 100 μl extract as sample. The Qia-shredder option significantly increases yield and purity of nucleic acid (see Qiagen instructions booklet).
6. Elute in 100 μl nuclease-free water.
7. Determine nucleic acid concentration and purity (see section 8.4.; “Nucleic acid quality assessment”).  
8. Store as two separate 50 μl aliquots at -80oC, one for working with and one for storage.
9. Include a ‘blank’ extraction (i.e. an extraction of purified water) after every 24 bee samples, to make sure none of the extraction reagents have become contaminated.