1.3.4. Chemical stabilizers
There are a number of chemicals that can be used to help stabilize nucleic acids during transport. Their purpose is to inhibit nucleases, especially the resilient RNAses, and in doing so destroy all enzymatic activity in the sample. So, if the final assays include natural enzymatic activity, these stabilizers should be avoided. For the similar reasons, many stabilizers are also incompatible with serological detection methods, such as ELISA.
A large excess (5-fold by
weight) of stabilizer should be added to ensure a high enough concentration
within the tissues for inhibiting RNAses. It is also essential that the
solution penetrates the tissues completely to abolish all RNAse activity. This
is a major difficulty for aqueous stabilizers, which cannot penetrate the
hydrophobic insect exoskeleton. These are therefore only suitable for extracted
tissues, eggs and small larvae, unless bodies are partially disrupted at the
start. Organic preservatives, such as 100% ethanol, have much more effective
penetration of the exoskeleton and are therefore better for stabilizing whole
adult bee samples. Although 100% ethanol is suitable for preserving RNA
destined for short-fragment RT-qPCR-based assays, storage in 70% ethanol has
been shown to result in strong degradation (Chen et al., 2007). However, recent data using
a short amplicon (124 bp) diagnostic for Deformed wing virus (DWV) in a Taqman
assay (Chantawannakul et al., 2006), showed no loss of DWV signal after adult
bees were stored for 4 weeks in 70% EtOH at room temperature compared to snap
frozen controls (G. Budge, unpublished data). RNA can also be stabilized
by high concentration sulphate salt solutions (Mutter et al., 2004), of which RNAlater® (Qiagen) is the best
known. A generic version can be made as follows:
700g di-ammonium sulfate
40ml 0.5M EDTA (pH 8.0)
25ml 1M tri-sodium citrate (di-hydrate salt; 29.4g/100ml)
1l sterile water
~1.3 l total volume
Once stabilized, RNase activities will be inhibited and samples can be stored for up to 1 month at 4°C, and long-term at -20°C or -80 ºC with minimal degradation. The stabilizer should be removed from the bee sample prior to homogenization and RNA extraction.
1. 100% ethanol
- Samples: whole adult bees; pupae; large larvae; tissues.
- Use: 5 volumes by weight.
- Storage: 1 month at room temperature or lower.
- Processing: Remove ethanol and process samples as normal.
- Pros: Cheap; effective penetration.
- Cons: Evaporation; possible transport restrictions;
heavy; incompatible with serological assays .
2. RNAlater® & generic equivalent
- Samples: tissues; eggs; small larvae.
- Use: 5 volumes by weight.
- Storage: 1 month at room temperature, or lower.
- Processing: Remove stabilizer and process samples as normal.
- Pros: Non-hazardous; effective penetration.
- Cons: Expensive (except generic version); heavy.
It is possible to use RNAlater® for darkened pupae and adult bees, if they are crushed into a paste or cut into 5mm sections (Chen et al., 2007). This is laborious and risks losing virus particles and RNA to the stabilizing solution, but may be required in certain circumstances. In such cases, the crushed bees should be centrifuged at 1,000rpm for 5 minutes at 4oC before removing the stabilizer and processing the crushed bee tissues.