Comparison of RNA extraction methods for RT-PCR detection of Coconut cadang-cadang viroid variant in orange spotting oil palm leaves

Figures & data

Fig. 1 (Colour online) (A) Oil palm seen with typical symptoms of orange spotting with severe crown discolouration. (B) A closer view of the affected fronds with orange spotting symptoms. (Figures courtesy of Malaysian Palm Oil Board.)

Table 1. Comparison of the different RNA extraction methods used in this study.

Extraction method	Optical density (OD) 260/280 (nm)	Optical density (OD) 260/230 (nm)	RNA concentration (ng µL^−1)	Amount of material used (g)	Processing time (h/day)
CTAB	1.80	2.00	2935.8	2	4 h/1 day
NETME	1.48	0.61	2717.6	2–5	6 h/2 days
PEG	1.11	1.02	240.2	20	3 days

Fig. 2 RT-PCR product from symptomatic samples extracted using the CTAB extraction and NETME methods. Lane M: 100 bp ladder; Lanes 1–3: symptomatic samples extracted using CTAB; Lane 4: SB 346 extracted using CTAB; Lane 5–7: symptomatic tissues extracted using NETME; Lane 8: SB 346 extracted using NETME; Lane 9: negative control (no template).

Fig. 3 RT-PCR product from asymptomatic samples extracted using the CTAB extraction and NETME methods. Lane M: 100 bp ladder; Lanes 1–3: asymptomatic samples extracted using CTAB; Lane 4: SB 346 extracted using CTAB; Lane 5–7: asymptomatic sample extracted using NETME; Lane 8: SB 346 extracted using NETME; Lane 9: negative control (no template).

Fig. 4 Secondary structure of CCCVd237 generated by Mfold. T_L: terminal left region; P: pathogenic region; C: central conserved region; V: variable region and T_R: terminal right.

Fig. 5 (Colour online) The phylogenetic tree of viroids constructed by MEGA 4 using the neighbour-joining method with 1000 bootstrap replicates. The bar indicates an evolutionary distance of 0.05%. Oil Palm OS (GenBank accession no. KT964100) derived from this experiment is boxed.