4.2. Affinity column purification

4.2. Affinity column purification

The processing consists of making a primary homogenate from 1-30 bees, purifying RNA from one (or more) aliquots of the homogenate using affinity columns, and measuring the RNA concentration. β-mercaptoethanol is toxic so processing should be done in a fume hood. Prepare all the buffers and tubes before starting.

The protocol below is based on the columns marketed by Qiagen or generic equivalents. The maximum recommended amount of tissue per column is 20 mg. More than 20 mg tissue causes the column to bind too much protein, reducing the yield and quality of the nucleic acid. Bees, pupae and large larvae weigh between 100-180 mg each, and so need to be homogenised first in a primary extract, from which a volume equivalent to 20 mg tissue is then processed on the affinity columns. A suitable denaturing buffer for this primary extract is a Guanidine Iso-Thio Cyanate (GITC) buffer, which has similar properties to the Qiagen RLT buffer:

1. Mix the GITC buffer:
5.25 M guanidinium thiocyanate (guanidine isothiocyanate),
50 mM TRIS.Cl(pH 6.4),
20 mM EDTA,
1.3% Triton X-100,
1% β-mercaptoethanol.

2. Place exact, pre-determined number of frozen bees in the homogenizer of choice.

3. Per bee, add the following amount of GITC buffer:

Bee

Weight    

Buffer    

Total volume    

Worker bee

120 mg

500 μl

600 μl

Drone

180 mg

700 μl

900 μl

Worker pupa    

160 mg

650 μl

800 μl

Drone pupa

240 mg

1000 μl

1200 μl

With these extract volumes, 100 μl extract is approximately 20 mg bee tissue

4. Mix:
100 μl bee extract,
350 μl RLT buffer + 1% β-mercaptoethanol.

5. Proceed according to the Plant RNA extraction protocol (see Qiagen instructions booklet).
Inclusion of the Qia-shredder column step is not required, but significantly increases yield and purity of nucleic acid.

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.

The BEEBOOK