Alternative RNA Extraction Method in Vibrio vulnificus Infected Brown- Marbled Grouper, Epinephelus fuscoguttatus

Publication date: 18 July 2019 To cite this article: Fahmeeda Mohamad Jazamuddin, Wan Mohd Aizat, Hoe-Han Goh, Chen-Fei Low and Syarul Nataqain Baharum. (2019). Alternative RNA Extraction Method in Vibrio vulnificus Infected Brown-Marbled Grouper, Epinephelus fuscoguttatus. Tropical Life Sciences Research 30(2): 201–209. https://doi.org/10.21315/tlsr2019.30.2.15 To link to this article: https://doi.org/10.21315/tlsr2019.30.2.15

Abstract: Vibriosis is a prevalent aquatic disease caused by Vibrio species and has led to massive loss of brown-marbled grouper, Epinephelus fuscoguttatus. The complexity of molecular mechanisms associated with immune defence can be studied through transcriptomics analysis. High quality and quantity of total RNAs are crucial for the veracity of RNA sequencing and gene expression analysis. A low quality RNA will compromise downstream analysis, resulting in loss of time and revenue to re-acquire the data again. Thus, a reliable and an efficient RNA isolation method is the first and most important step to obtain high quality RNA for gene expression studies. There are many aspects need to be considered when deciding an extraction method, such as the cost-effectiveness of the protocol, the duration of chemical exposure, the duration required for a complete extraction and the number of sample-transferring. A good RNA extraction protocol must be able to produce high yield and purity of RNA free from enzyme inhibitors, such as nucleases (RNase), phenols, alcohols or other chemicals carryover, apart from protein and genomic DNA contamination, to maintain isolated RNA integrity in storage condition. In this study, TransZol TM Up produced clean and pure RNA samples from control gills only but not from the infected gill and whole-body tissues. Modified conventional CTAB (conventional hexadecyltrimethylammonium bromide) method was then used as an alternative method to isolate RNA from gill and whole-body tissues of Vibrio-infected E. fuscoguttatus. Modified CTAB method produced intact RNA on gel electrophoresis with higher RIN number (>6.5) for infected gill and whole-body tissues, suggesting that this method could also be used to isolate high quality RNA from fish samples. Therefore, this method is potentially suitable to be used to extract RNA from other fish species especially those that have been infected.
Keywords: CTAB, Gill, RNA Extraction, TransZol TM Up, Whole-Body Tissues Vibriosis is a type of systemic disease (Chatterjee & Haldar 2012) caused by the Vibrio species, a Gram-negative bacteria which can cause severe illnesses to certain internal organs of the infected hosts leading to death (Chen et al. 2000;Geng et al. 2014). It often attacks grouper cultures including brown-marbled grouper (Epinephelus fuscoguttatus) causing the grouper production started to decrease gradually (Ilmiah et al. 2013). Information on host-bacteria interactions and the immune responses underlying E. fuscoguttatus and vibriosis are still largely unknown. Transcriptomic profiling analysis has been extensively applied in fish studies for the discovery of many immune-related genes and is a very useful technique in revealing the genetic response of host towards pathogen and disease infections (Ali et al. 2014;Huang et al. 2011;Qi et al. 2016;Wang et al. 2016). Thus, to achieve an overall success of gene expression analyses, a reliable and an efficient RNA isolation method is the first and most important step to obtain high quality RNA for gene expression studies (Bharudin et al. 2014). TriZol reagent, a monophasic solution of phenol and guanidinium isothiocyanate (Rio et al. 2010), is one of the most common technique widely used for extracting RNA from fish samples (Ali et al. 2014;Cui et al. 2011;Lee et al. 2016;Li et al. 2016;Rajan et al. 2013). Meanwhile, conventional hexadecyltrimethylammonium bromide (CTAB) is a non-ionic detergent that is very convenient for the isolation of DNA and RNA from polysaccharides-rich organisms such as plants (Tan & Yiap 2009). Although CTAB-based method is the major nucleic acids extraction method used for plant leaf tissues (Healey et al. 2014), its usage for extracting RNA from other types of samples such as fish is yet to be explored. To our knowledge, there is currently no studies reported on extracting RNA from fish samples specifically from fish wholebody or other any organs such as gill by using CTAB method.
Fish sampling were conducted at Makmal Penyakit Ikan, Universiti Malaysia Terengganu (UMT). A total of 300 brown-marbled groupers (Epinephelus fuscoguttatus) fingerlings with average length of 5 cm and average weight of 2.20 g obtained from Fisheries Research Institute (FRI) Tanjung Demong, Besut in Terengganu, Malaysia were acclimatised in six glass aquariums (50 fishes/aquarium) with running aerated seawater (25°C, salinity ~35 parts per thousand (PPT), pH 7.9) for one week. For V. vulnificus experiment, 150 healthy grouper fingerlings were immersed in 20 L seawater containing 1 x 10 7 CFU mL −1 concentration of Vibrio vulnificus for 30 min; whereas for control another 150 fingerlings were immersed in clean seawater, before transferred into a new aquarium containing new seawater, and observed daily for 30 days. At day 31-post-infection, survived fish of control and infected groupers were collected at the same time point and stored separately in -80°C freezer until RNA extraction. Then, RNA were firstly extracted from gill and whole-body tissues using TransZol TM Up Plus RNA kit (Nanogene Solution, TransGen Biotech, Beijing, China) following the manufacturer's protocols. Total RNA extracted were then treated with DNase I (RNase-free) (Thermo Fisher Scientific Inc.) to remove DNA contamination. The second method used was modified from CTAB RNA extraction method by Kim and Hamada (2005) as well as Abdul-Rahman et al. (2017). Ground gill tissues (30 mg) and whole-body tissues (2.5 g) of the fish were transferred to tubes containing 1 mL and 25 mL of CTAB extraction buffer (0.5 mM pH 8.0, 1 M Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 8.0, 2M sodium chloride (NaCl), 2% hexadecyltrimethyl ammonium bromide (CTAB) (w/v), 2.5% polyvinylpyrrolidone 40 (PVP-40) (w/v) and 2% β-mercaptoethanol), mixed gently and incubated at room temperature for 3 min and 5 min, respectively. An equal volume of chloroform/isoamyl alcohol (24:1 v/v) was added to each sample and completely mixed by inverting the tubes gently. The solutions were centrifuged at 13,000 rpm at 4°C for 20 min. Supernatants formed were transferred into new tubes and the previous step was repeated once before adding 1/3 of 12 M lithium chloride. Samples were precipitated by incubation at -20°C for 3 h. Then, solutions were centrifuged at 13,000 rpm for 90 min for whole body tissues, and 60 min for gill tissues at 4°C. A total of 1.0 mL of 0.1% DEPC-water was added to resuspend the precipitated samples. An equal volume of chloroform/isoamyl alcohol (24:1 v/v) was added, mixed gently, and centrifuged at 13,000 rpm for 20 min. Supernatants were transferred to new tubes and added with 1/3 of 12 M lithium chloride before kept at -20°C for 3 h. Solutions were then centrifuged (13,000 rpm, 30 min, 4°C) and pellets were washed twice with 70% ethanol. The RNA pellets were airdried for 2 min and resuspended in 0.1% DEPC-water without DNase treatment. Compared to the extraction of RNA from mangosteen pericarp by Abdul-Rahman et al. (2017), the extraction of RNA from gill and whole-bod tissues differs in incubation condition and duration of the extraction. The current study's incubation time for lysing the animal tissues were 3-5 minutes at room temperature while the study conducted by Abdul-Rahman et al. (2017) incubated the sample at 65°C for an hour. Besides that, Abdul-Rahman et al. (2017) took three days to extract the RNA samples whereas the current study took only half day. The quality of total RNA isolated were then assessed based on absorbance ratio of A 260/280 and A 260/30 , agarose gel electrophoresis and RNA integrity number (RIN) score.
Based on Table 1 and Table 2, when compared to TransZol TM Up, CTAB extraction method produced a comparable amount of RNA concentration for both gill (214.52 ng/µL for control and 142.67 ng/µL for infected) and whole-body (149.81 ng/µL for control and 168.03 ng/µL for infected) tissues. Besides that, based on the absorbance ratios, the A 260 /A 280 for total RNA extracted using CTAB method also resulted with acceptable ratio when compared to TransZol TM Up indicated that CTAB method also can effectively remove contamination from the total RNA extracted. CTAB protocol can be advantageous compared to TransZol TM Up as it has more cleaning steps during the extraction process. This is because, CTAB protocol includes liquid-liquid phase separation step that needed to be done three times during extraction process. Liquid-liquid phase separation step is the addition of chloroform with isoamyl alcohol to the extracted solution. This step is very critical because it ensures higher cleaning and removing of DNA, protein and any contaminants in the supernatant (Tan & Yiap 2009) providing the samples three times cleaner. As for TransZol TM Up kit, it only requires one time cleaning step, which is only the addition of chloroform for phase separation. Next, the gel electrophoresis results shown in Fig. 1 are the images of 18S and 28S rRNA bands for gills and whole-body tissues after extraction by TransZol TM Up kit with DNase treatment and CTAB method without DNase treatment. The results suggested clear and intact 18S and 28S rRNA bands of gill (Figure 1(a)) and whole-body (Fig. 1b) for extraction using CTAB method when compared to TransZol TM Up. RNA samples extracted using CTAB did not show visible genomic DNA contamination even without DNase treatment because apart from having better liquid-liquid phase separation, CTAB also has better RNA precipitation step that is by using lithium chloride (LiCl). LiCl precipitation provides a major advantage over other precipitation methods because it can efficiently precipitate RNA but does not effectively precipitate DNA, proteins, and carbohydrates making it most suitable for purification of RNA (Walker & Lorsch 2013).
Apart from that, the quality of total RNA extracted from gill and whole-body tissues were analysed by Agilent 2100 Bioanalyzer to determine RNA Integrity Number (RIN). The corresponding electropherograms for gill and whole-body samples were shown in Fig. 2. Based on the RIN score, TransZol TM Up produced clean and pure RNA samples from the control gill (RIN more than 7) only but not from the infected gill (Table 1) and whole-body tissues (Table 2). Extracting RNA from the infected gill and whole-body tissues could be challenging for TransZol TM Up due to the pathological effects caused by V. vulnificus infection such as hyperproduction of mucus, skin discolouration, fin erosion, skin lesion, haemorrhage and reddening of the internal organs including gill. These effects might cause additional contamination to the samples. Contamination by other organisms and excessive production of mucus are some of the challenges associated with the isolation of   (Huang et al. 2012) and mangosteen pericarp (Abdul-Rahman et al. 2017). Thus, this suggested that the effectiveness of CTAB protocol showed that CTAB method can be an alternative method for extraction of RNA from diseased tissues including fish samples. In conclusion, the evaluation of two RNA extraction methods showed that TransZol TM Up is suitable for extraction of RNA from uninfected (control) gill while CTAB method is preferred for the extraction from infected gill and whole-body tissues. High purity of total RNA, intactness of rRNA bands and higher RIN score from CTAB method was also considered to be suitable for extracting RNA from difficult tissues especially animal tissues that are greatly infected by disease.