Abstract
Caenorhabditis elegans is a well-established laboratory animal model and has been widely used in biological research. However, it is still a challenge to obtain a good amount of quality RNA from a limited number of C. elegans for gene expression studies. To address this issue, the present study has compared different conditions to preserve C. elegans for RNA extraction after the failure of an initial effort to use RNAlater-preserved worms for RNA extraction. The effects of different concentrations of proteinase K, different worm life stages, and different worm numbers on RNA extraction were also investigated. The best results were achieved under the following conditions: 1) adult worms that were either freshly prepared or quickly frozen in liquid nitrogen followed by storage at −80 °C; 2) disruption of C. elegans with proteinase K (1 mg/mL) in a lysis buffer (65 °C for 10 min) prior to extraction with Trizol agent. This method can provide a stable, rapid, and effective means to extract RNA from C. elegans with variable worm numbers from 20 to 200.•RNAlater was inappropriate for preserving C. elegans for effective RNA extraction.•Proteinase K was verified for lysing a limited number of C. elegans for RNA extraction.
Highlights
Method ArticleInappropriateness of RNAlater to preserve Caenorhabditis elegans for RNA extraction Leming Jianga,b, Linyan Lia,b, Ping Kangb,c, Hai Yub, Shao-Ping Niea, Ming-Yong Xiea, Joshua Gongb,*
Concentrations on the lysis of C. elegans, worms were firstly collected into a 15 mL centrifuge tube using RNAse free water, vortex and centrifuged at 300 rpm to remove the supernatant, repeated it three times to wash the worms. 20 washed worms were transferred in 5 mL RNase-free water to a new 200 mL RNase-free Eppendorf tube, mixed with 20 mL of basic lysis buffer containing 1 or 2 mg/mL of proteinase K (Qiagen, Cat.#19133), and incubated at 65 C for 2, 5, or 10 min followed by 85 C for 1 min
Statistical significance of the data was performed by using one-way analysis of variance (ANOVA) in Statistical Product and Service Solutions (SPSS, Windows version 11.5), and a value of P 0.05 was accepted to be statistically significant
Summary
Inappropriateness of RNAlater to preserve Caenorhabditis elegans for RNA extraction Leming Jianga,b, Linyan Lia,b, Ping Kangb,c, Hai Yub, Shao-Ping Niea, Ming-Yong Xiea, Joshua Gongb,*. A State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi, 330047, China b Guelph Research and Development Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, N1G 5C9, Canada c Hubei Key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan, 430023, China
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