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. 2015 Apr;77(4):461-5.
doi: 10.1292/jvms.14-0227. Epub 2014 Dec 27.

Establishing conditions for the storage and elution of rabies virus RNA using FTA(®) cards

Takeo Sakai  1 , Ayako Ishii, Takao Segawa, Yukihiko Takagi, Yuki Kobayashi, Takuya Itou
Affiliations

Establishing conditions for the storage and elution of rabies virus RNA using FTA(®) cards

Takeo Sakai et al. J Vet Med Sci. 2015 Apr.

Abstract

The Flinders Technology Associates filter paper cards (FTA(®) cards) can be used to store nucleic acid from various samples and are easily portable. However, RNA is physicochemically unstable compared with DNA, and appropriate methods have not been established for storage and extraction of RNA from FTA(®) cards. The present study investigated the optimum conditions for storage and elution of viral RNA (vRNA) using rabies virus (RABV) applied to FTA(®) cards. When TE buffer was used, the elution rates of vRNA increased with the length of the elution time. When the cards were stored at -80 °C or -20 °C, vRNA was stable over 3 months. Degradation of vRNAs occurred following storage at 4 °C and room temperature, suggesting that RNA should be extracted from cards as soon as possible if no freezer is available. When we tried to amplify vRNA from RABV-infected animal brains applied to FTA(®) cards and stored at -80 °C for 6 months, we did not detect any amplified products with the primer set for 964 bp of RABV N gene. However, we were able to detect amplified products by increasing the elution time of vRNA from FTA(®) cards from 30 min to 24 hr or by changing the primer sets to amplify 290 bp of N gene. Thus, we recommend extending the elution time for damaged or low concentration samples in FTA(®) cards.

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Figures

Fig. 1.
Fig. 1.
Comparison of elution rates of vRNA from FTA® cards using five different eluents. The symbols and vertical bars indicate the average values and standard deviations of extraction rates of vRNA from the FTA® card disks. Extraction rates of vRNA were estimated from the copy numbers of RNA by real-time PCR with the primer pairs of realNgeneF1 and realNgeneR1.
Fig. 2.
Fig. 2.
Influence of storage temperatures and time periods of FTA® cards on vRNA yield from the cards. The graphs indicate the average values with standard deviations calculated from the extraction amounts of vRNAs from the FTA® card disks. *P<0.01.
Fig. 3.
Fig. 3.
vRNA yield from FTA® cards with and without mouse brain emulsion. The FTA® cards with samples were stored at −80°C. The graphs indicate the average values with standard deviations calculated from the extraction amounts of vRNAs from the FTA® card disks.
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