Evaluation and Standardization of RNA Extractions with Quality for RNA-Seq for Balamuthia mandrillaris

Abstract

Balamuthia mandrillaris is a free-living amoeba (FLA) that causes granulomatous amebic encephalitis (GAE) and skin lesions. Transcriptomic analysis is a powerful tool used to study B. mandrillaris pathogenic infections. However, preliminary tests of RNA extraction showed poor results, so it has become essential to standardize a protocol for high-quality RNA. The present study evaluated 11 RNA extraction protocols based on three commercial kits by making modifications to the temperature and centrifugation times, and by combining kits. Four protocols, namely Q3 (based on QIAGEN RNeasy Mini Kit, with modifications in temperature and centrifugation times), T1 (Invitrogen TRIzol Reagent), T2 (combination of TRIzol and QIAGEN modified protocols) and T3 (combination of TRIzol and PROMEGA SV Total RNA Isolation protocols), presented RNA with good integrity and purity, except for the T1 protocol, which obtained an A260/230 value below the acceptable threshold. High RNA integrity (RIN) values were obtained with the Q3 (9.8), T2 (9.2), and T3 (8.9) protocols, while the T1 protocol obtained a lower RIN value (7.1). The Q3, T2, and T3 protocols obtained high-quality RNA from B. mandrillaris based on the criteria of integrity, purity, and concentration, where the implemented modifications and combinations raised the quality; thus, their use is recommended to obtain accurate results when performing transcriptomic analysis.

1. Introduction

Free-living amoebae (FLAs) are protozoa distributed worldwide and found in different environmental sources, such as soil and water [1]. Among the FLAs that have been reported as pathogenic for humans are the genera Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrillaris, Vermamoeba vermiformis and Sappinia pedata [2]. Balamuthia mandrillaris is a protozoan found primarily on soil samples, although in some cases it has been possible to isolate it from bodies of water [3]. This pathogen causes granulomatous amebic encephalitis (GAE), which affects the central nervous system (CNS), and causes skin lesions in infected people. Currently, more than 200 cases of infection by this FLA have been diagnosed around the world, with more than 100 cases reported in America [4,5]. GAE can reach a mortality rate > 95%, and although there is no 100% effective treatment, the use of certain antibiotics and antifungals combined with an early diagnosis can increase the chances of survival for the infected [6].

To study the pathogenesis of the infections caused by B. mandrillaris, transcriptomic analyses can be carried out to identify and quantify the set of transcripts in a cell at a specific stage or condition that could be involved in the development of the disease [7,8]. The information on B. mandrillaris in the transcriptomic analysis is sparse. In 2021, functional annotation was carried out on 40% of the sequences corresponding to the proteome [9], and in 2023, the functional annotation and comparative genomic analysis of this amoeba revealed potential virulence-related genes [10]. To perform transcriptomic analysis, an extraction protocol for B. mandrillaris that provides high-quality ribonucleic acid (RNA) is necessary, considering its integrity, purity, and quantity [11]. In preliminary tests, we found problems in the performance of high-quality RNA extraction using commercial kits and following the manufacturer’s instructions; these difficulties could be due to certain factors such as the B. mandrillaris cyst, which is very resistant to both physical and chemical treatments, hindering its lysis and hence the extraction of nucleic acids [12,13]. This investigation aimed to obtain RNA from B. mandrillaris that complies with the integrity, purity, and quantity parameters required for its use in RNA-Seq analysis by standardizing the extraction protocol.

2. Results and Discussion

2.1. RNA Purity and Concentration

All samples were analyzed with Nanodrop 2000c equipment (

Table 1. Results regarding the concentration, purity and integrity of RNA extracted with different protocols.

Protocol	Concentration (ng/µL)	Final Quantity (µg)	A260/280	A260/230	Integrity (Gel Electrophoresis)
P1	322.78 ± 21.66 a	32.28 ± 2.16	2.20 ± 0.00 d	1.74 ± 0.04 abcd	✗
P2	334.55 ± 26.94 a	33.45 ± 2.62	2.01 ± 0.00 ab	2.47 ± 0.01 de	✗
P3	125.00 ± 3.74 a	12.50 ± 0.37	2.13 ± 0.00 cd	1.30 ± 0.03 a	✗
Q1	284.15 ± 15.43 a	14.21 ± 0.77	2.11 ± 0.02 bcd	1.54 ± 0.14 abc	✗
Q2	380.80 ± 21.90 ab	19.04 ± 1.09	2.11 ± 0.01 bcd	2.22 ± 0.02 bcde	✗
Q3	1454.77 ± 37.49 d	72.74 ± 1.87	2.11 ± 0.01 bcd	2.29 ± 0.02 cde	✓
Q4	690.08 ± 37.44 bc	34.50 ± 1.87	2.15 ± 0.00 cd	2.39 ± 0.02 de	✗
Q5	342.12 ± 14.08 a	17.11 ± 0.70	2.05 ± 0.00 abc	2.25 ± 0.01 cde	✗
T1	852.61 ± 91.04 c	42.63 ± 4.70	1.98 ± 0.02 a	1.46 ± 0.13 ab	✓
T2	818.25 ± 11.45 c	40.91 ± 0.57	2.15 ± 0.00 cd	2.59 ± 0.00 e	✓
T3	795.28 ± 31.30 c	79.53 ± 3.13	2.22 ± 0.00 d	2.40 ± 0.01 de	✓

✓: Integral sample, ✗: Non-integral sample. Determined by the intensity of the RNA bands and the 2:1 ratio of the 28S subunit to the 18S subunit. Values with different letters are significantly different (p < 0.05).

), beginning with the protocols following the manufacturer’s instructions. The concentration values obtained from protocols P1 (322.78 ± 21.66 ng/µL), Q1 (284.15 ± 15.49 ng/µL) and T1 (885.9 ± 94.05 ng/µL) were acceptable, but due to non-optimal purity values, it was decided that the temperature (from 20–25 °C to 4 °C) and centrifugation time (from 1 to 3 min for PROMEGA protocols and from 15 s to 1 min for QIAGEN protocols) would be modified, and an additional precipitation step performed. It is well known that RNA is too unstable at room temperature due to its structure, and one way to prevent degradation is to work at low temperatures [14]. Ethanol precipitation is able to separate the RNA molecules from the solution, and can be used to obtain a sample with a good purity and concentration [15].

With the modifications applied, an improvement in purity was noticed; the Q3 protocol had the best performance, with an average concentration of 1454.77 ± 37.49 ng/µL, and values for the 260/280 ratio of 2.1 ± 0.01 and values for the 260/230 ratio of 2.2 ± 0.02. The T2 and T3 protocols also presented an improvement.

Although the T3 protocol concentration is lower than that of the T1 and T2 protocols, the final quantity is greater due to the difference in the elution volumes (T3: 100 µL, T1 and T2: 50 µL). The T1 protocol has a slightly higher concentration than the T2 protocol, but an improvement was demonstrated in the A260/230 ratio, and a higher RIN value (

Table 2. RNA Integrity Number (RIN) analysis of selected protocols.

RNA Sample	RNA Integrity Number (RIN)
T1	7.1
Q3	9.8
T2	9.2
T3	8.9

).

The RNA concentration is important for transcriptomic analysis (>2 µg). Low concentration levels affect the amount of library data available in a sequencing process, making it more likely to present amplification artifacts that could affect the interpretation of the results [16]. In RNA, absorbance at A260 primarily measures nucleic acids, indicating the RNA concentration; absorbance at A280 detects proteins; and absorbance at A230 identifies organic compounds. Also, RNA has a higher 260/280 ratio due to the ratio of Uracil (4.00) compared to that of Thymine (1.47) [17]. This is validated through spectrophotometry using the 260/280 ratio, with an optimal value above 2.0 for RNA [18], and the 260/230 ratio, with an optimal value of 2.2 [19]. Values above 2.0 for the A260/280 ratio indicate the absence of protein contamination, and values above 2.0–2.2 for the A260/230 ratio indicates the absence of solvent contamination and denote the isolation of high-quality, non-contaminated, or pure RNA [17,20].

2.2. RNA Integrity

All samples were analyzed by gel electrophoresis. Only protocols Q3 (Figure 1A), T1 (Figure 1B), T2 (Figure 1C), T3 (Figure 1D) showed good integrity based on the intensity and ratio of the 28S and 18S rRNA bands.

RNA is considered of high quality when the 28S subunit has a 2:1 ratio over the 18S subunit in gel electrophoresis [21,22,23], so T1, Q3, T2 and T3 comply with this characteristic. The RNA integrity in electrophoresis gel is evidenced by the presence of 2 kb and 4 kb bands, which indicates that the RNA is not degraded; this is a critical quality control step for experiments such as transcriptome sequencing [24].

Likewise, the decrease in the centrifugation temperature to 4 °C had a positive effect on the integrity of the modified protocols, combined with the use of DEPC water for the agarose gel preparation and electrophoresis; this is due to its inhibition of RNase activity, since in preliminary tests, electrophoresis gels were used without DEPC water, resulting in RNA degradation in all samples. RNA integrity analysis is used to determine the level of degradation that a sample presents; its evaluation is important to obtain reliable results in experiments that involve the study of the transcriptome [25].

2.3. Determination of RIN

The protocols that showed good integrity during gel electrophoresis were analyzed by chip electrophoresis using a 2100 Bioanalyzer system (Agilent, Santa Clara, CA, USA) to obtain the RIN value (Table 2). The T1, Q3, T2, and T3 protocols obtained RIN values of 7.1, 9.8, 9.2, and 8.9, respectively. The RIN determines the RNA integrity of a sample, with values ranging from 1 to 10 (10 being the highest quality), and an RIN with a value ≥ 8 is considered to be of suitable quality. The RIN is calculated based on an electrophoretic trace with an algorithm that evaluates the ratio of ribosomal RNA (rRNA) peaks, specifically the 18S and 28S rRNA bands, which are prominent in high-quality RNA [26,27,28]. The T1 protocol exhibited an RIN of 7.1, the lowest compared to the other protocols; this may be due to the purity levels because it does not use an RNA extraction protocol that includes washing through columns.

3. Materials and Methods

3.1. Culture and Harvest of B. mandrillaris

Balamuthia mandrillaris ITSON (BMI) medium was used for the axenic culture of the B. mandrillaris ITSON01 strain. This medium is composed of 10 g of Bacto™ Casitone (Difco, Detroit, MI, USA) in a final volume of 500 mL of ddH₂O sterilized by an autoclave, and 10% (v/v) fetal bovine serum, 200 UI/mL of penicillin, and 200 μg/mL of streptomycin as antibiotics; this was alongside a complement of 34 mL of Hank 10X balanced salt solution, which is composed of the following: CaCl₂ 144 mg/L, MgCl₂.6H₂O 1000 mg/L, MgSO₄.7H₂O 1000 mg/L, KCl 4000 mg/L, KH₂PO₄ 600 mg/L, NaCl 80,000 mg/L, Na₂HPO₄.7H₂O 900 mg/L and D-glucose (Dextrose) 10,000 mg/L. [29]. For harvesting, 75 cm² cell culture flasks with 9 mL of BMI medium and 1 mL of fetal bovine serum were incubated at 37 °C for one week. The samples were refrigerated at 4 °C for 10 min before they were resuspended (since B. mandrillaris adheres to the surface and this thermal shock seems to help to detach amoebas that have this characteristic [29,30]); this was followed by two steps of washing using phosphate-buffered saline (PBS, pH 7.4) (except in protocols employing TRIzol, where only cell harvesting was performed) and centrifugation at 4000 times gravity (×g) for 10 min.

3.2. RNA Extraction Protocols

Three different commercial kits were used to carry out the RNA extraction protocols, but due to modifications and combinations, a total of 11 protocols were evaluated: the PROMEGA SV Total RNA Isolation System, QIAGEN RNeasy Mini Kit and Invitrogen TRIzol Reagent were used, following the manufacturer’s instructions; then, the temperature and centrifugation times were modified and a precipitation step was added once the RNA was eluted.

3.2.1. PROMEGA SV Total RNA Isolation System

We performed three different protocols based on this commercial kit: the first followed the manufacturer’s instructions (P1) (Figure 2), and the second protocol (P2) followed the same methodology (Figure 2) but added a precipitation stage once the RNA was eluted, using 10% sodium acetate (3M) of the total volume of eluted RNA, and 3 volumes (total eluted RNA) of cold molecular biology-grade absolute ethanol. Samples were refrigerated at −70 °C for 24 h, and then centrifuged at 20,000× g and 4 °C for 30 min. The pellet was washed twice, with 500 μL of 75% ethanol, and centrifuged at 20,000× g for 10 min. The supernatant was removed, and the pellet was allowed to dry for 8 min. Finally, the pellet was resuspended in nuclease-free water according to the final volume required and stored at −70 °C [15]. In the last protocol (P3) (Figure 2), changes were made to the centrifugation temperature, being altered from 20–25 °C to 4 °C, and the speed was set to 14,000× g and the time set from 1 min to 3 min.

3.2.2. QIAGEN RNeasy Mini Kit

We carried out five different protocols based on the QIAGEN RNeasy Mini Kit: the first followed the manufacturer’s instructions (Q1) (Figure 3), and the second protocol (Q2) followed the same methodology but included the same additional precipitation once the RNA was eluted, as described for the P2 protocol. We made changes to the centrifugation for the third protocol (Q3) (Figure 3), changing the temperature from 20–25 °C to 4 °C and the time from 15 s to 1 min. The fourth protocol (Q4) followed the same methodology as the Q3 protocol but added a treatment with DNase I (described in the manufacturer’s instructions) (Figure 3). For the last protocol based on this kit (Q5), the same methodology used in the Q3 protocol was applied, alongside the additional precipitation described for the P2 protocol (Figure 3).

3.2.3. Invitrogen TRIzol Reagent

Three different methodologies were implemented for the RNA extractions based on the Invitrogen TRIzol Reagent kit: the first followed the manufacturer’s instructions (T1) (Figure 4); the second combined the Invitrogen TRIzol Reagent lysis procedure and the QIAGEN RNeasy Mini kit, with centrifugation and temperature modifications (T2) (Figure 5); and the third combined the Invitrogen TRIzol Reagent lysis procedure and the PROMEGA SV Total RNA Isolation System (T3) (Figure 5).

3.3. Analysis of RNA Purity, Concentration, and Integrity

All RNA samples were analyzed by spectrophotometry using Nanodrop 2000 c equipment to determine the purity and concentration. The RNA integrity was determined by agarose gel electrophoresis. The RNA integrity number (RIN) values were determined with chip electrophoresis using a 2100 Bioanalyzer system (Agilent, Santa Clara, CA, USA).

3.4. Gel Electrophoresis

As a previous step, the following reagents were prepared:

DEPC water: 1 L of distilled water and 0.1% of the final volume of DEPC (diethyl pyrocarbonate) were homogenized, incubated at 37 °C for 2 h, and sterilized at 120 °C for 15 min.

TBE (Tris-borate-EDTA) buffer 5×, 27 g of Tris-base, 13.75 g of boric acid and 10 mL of 0.5 M EDTA (ethylenediaminetetraacetic acid) were mixed in 350 mL of DEPC water. After the contents were dissolved, the pH was adjusted to 8 using KOH beads, and the volume was adjusted to 500 mL. From this solution, a 0.5× dilution was made to prepare agarose gels and electrophoresis runs.

The agarose gels were prepared with 40 mL of TBE 0.5× and 0.4 g of agarose, and ran at 90 v for 35 min. These were visualized using a Bio-Rad Gel Doc XR + Molecular Imager transilluminator.

3.5. Chip Electrophoresis

Chip electrophoresis was performed to determine the RNA integrity and obtain the RIN, just for those protocols that showed good integrity during gel electrophoresis. The RIN was calculated using the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) following the chip preparation methodology provided by the manufacturer, with the Eukaryote Total RNA Nano Series II assay performed using the Agilent 2100 Expert Software program.

3.6. Statistical Analysis

Eleven RNA extraction protocols were carried out with five repetitions (n = 5), implementing different combinations and modifications based on three commercial kits; in total, 55 samples were obtained, and these were analyzed to determine the purity, concentration, and integrity. Statistical analyses were performed using Statgraphics Centurion XVI software, using one-way ANOVA and a post hoc comparison of means with the Tukey HSD test. The factors were the RNA extraction protocols, and the response variables were the RNA concentration, 260/280 and 260/230 ratios.

4. Conclusions

In conclusion, through the harvest of B. mandrillaris in cell culture bottles, a sufficient RNA concentration was obtained for the evaluation of the RNA extraction protocols based on three different commercial kits and methodologies, which allowed the performance of each to be analyzed based on the quality, integrity, and quantity of the extracted RNA, as well as those samples suitable for RNA-Seq analysis to be determined. The modifications that were made to the temperature parameters and centrifugation times, as well as the combination of protocols, positively affected the final RNA quality, maintaining the integrity and increasing the purity. The RIN is a crucial metric used to assess the integrity of RNA, and the RIN values we achieved confirm that the RNA was largely intact and free from significant degradation, making it highly suitable for downstream applications. In addition, working with the optimal conditions for the equipment and materials and using reagents such as DEPC water are important for the correct functioning of the RNA extraction protocols.

Therefore, based on the RIN values (indicating highly intact RNA samples), integrity analysis, and concentration and purity metrics, it is recommended that protocols Q3, T2, and T3 are used to obtain high-quality RNA and accurate and precise results when performing transcriptomic analysis.