1. Introduction
Tuberculosis remains a major global health threat and affects about one third of the global population with estimated incidents of 10 million new cases and deaths of 1.2 million people in the year 2018. In the same year, the global report of drug-resistant tuberculosis was also about half a million (417,000 to 556,000 people), in which 3.4% of the new cases and 18% of previously treated cases had the chance to develop multidrug-resistant tuberculosis (MDRTB). Hence, the development of new and novel diagnostic tools is one of the pillars to curb this challenge [
1]. Depending on the methods utilized, diagnosis of tuberculosis has various shortcomings. Low sensitivity and specificity, delay in diagnosis, difficulty in diagnosis of child and extrapulmonary tuberculosis, lack of early detection of treatment respondents and non-respondents, and absence of accurate disease progression markers are some of the diagnostic challenges [
2]. Thus, improving tuberculosis diagnosis though new and innovative methods is needed to enhance tuberculosis control and management.
Currently, various molecular markers have been developed and implemented to increase tuberculosis diagnosis. Of these, miRNA is becoming a promising marker in diagnosis, as well as in prognosis for both infectious and non-infectious diseases [
3]. MicroRNAs (miRNAs) are short (~19 to 24 nucleotides in length) noncoding, evolutionary conserved RNAs that are broadly expressed in the genomes of animals and humans [
4,
5]. Earlier studies also reported that miRNA regulates gene expression at the post-transcriptional level and is involved in a wide range of cellular processes, including apoptosis, cell differentiation, and proliferations [
6].
MicroRNA synthesized in the immune and non-immune cells are secreted out and enters extracellular human body fluids, including blood plasma, urine, saliva, and semen. Such microRNAs circulate in the bodily fluids, and the blood stream reaches various parts of the body distal to the site of infection [
7,
8,
9]. MicroRNA in the extracellular environments have shown extreme stability in the fluids of mammals [
7,
10], at room temperature, and in adverse conditions, such as multiple freeze–thaw cycles and RNase degradation. The stability at varied conditions add to the regulatory role and differential expression in body fluids of health and disease’s state makes circulating miRNA a promising biomarker for diagnosis, prognosis, and treatment monitoring for various types of diseases, including tuberculosis [
11,
12].
The quantitative polymerase chain reaction (qPCR) is a widely applicable molecular technique that is used to quantify the expression of transcripts in certain biological specimens. The comparison between normal and diseased individuals with different groups requires a reference gene which is stable in all the groups. Hence, selection and normalization of the reference gene is a crucial step in qPCR experiments, which also affects the overall transcript level and its biological interpretations [
13].
Basically, constant expression in all target cells and study groups, stability in different conditions, and detectability are the key features of housekeeping genes [
14]. However, it has been reported that the stability of reference miRNA for studying circulating miRNA varies significantly among studies, implicating the lack of universal reference miRNA. Although miRNA-16-5p and RNU6B was widely accepted as an internal reference, miR-93, miR-22, miR-26a, miR-191, miR-320, and let-7i have also been reported as an endogenous control for circulating miRNA in tuberculosis and non-tuberculosis diseases [
15]. Thus, this study aimed to identify stable endogenous circulating miRNA that will be useful to normalize the genetic expression of plasma miRNA derived from tuberculosis-infected and noninfected health controls.
4. Discussion
MicroRNA is a class of small RNA (18 to 24 nucleotides) that play regulatory roles in gene expression at the post-transcriptional level, either through translational repression or mRNA degradation [
26,
27]. They regulate almost one-third of the known protein coding sequences, and are involved in various cellular processes, including cell proliferation, apoptosis, and signaling pathways [
4,
28]. The mature miRNAs that are found in varieties of bodily fluids, circulating miRNA, also regulate a wide range of processes, both in immune and non-immune cells, and affect their genes expression. Mounting evidence has reported that the level of circulating miRNA, up/down or deregulation are associated with specific physiological conditions. Thus, the variation in gene expression can be utilized as diagnostic and prognostic markers in many non-infectious and infectious diseases, including tuberculosis [
11,
29,
30,
31]. The stability in nature and various external conditions, expressions associated with changes in physiological conditions, abundance in bodily fluids, and noninvasiveness emphasize circulating miRNA as a source of stable biomarkers [
9,
10,
11,
32].
The quantitative polymerase chain reaction (qPCR) is the most powerful and common technique used for quantification of nucleic acid molecules that reflects the biology of tested samples. The data accuracy and quality that results from qPCR can be affected by several factors, including biological variability, sample storage conditions, nucleic acid extraction, cDNA synthesis, qPCR data computation, and reference gene (internal control) validation [
13]. However, the lack of universal endogenous controls for miRNA in biofluids remains an impediment for accurate analysis of circulating miRNAs’ expression. Therefore, the validation of an optimum endogenous control is a crucial step in qPCR experiment that ensures the reliability of data generated as well [
13]. In this study, we examined the suitability of six candidate reference genes: let-7i-5p, let-7a-5p, miRNA-16-5p, miRNA-22-3p, and miRNA-93-5p, as well as RNU6B to normalize the expression of target miRNA in plasma.
Although RNU6B and miR-16 are the most common reference genes utilized to normalize circulating miRNA expression, recent studies have reported that both are not stable markers (endogenous control) in serum and plasma samples of all disease types [
33,
34]. Thus, a wide range of miRNA has been reported as endogenous controls. Of these, miR-93-5p, let-7a, miR-221, miR-26a, miR-191, and miR-320a have been commonly identified stable circulating miRNAs for various types of diseases and pathological conditions [
15]. Our observation also confirmed that the expression of RNU6B is highly variable and was excluded from further data analysis, as its readings were above the threshold (data not shown).
We employed multiple algorithms, such as Best Keepers, geNorm, Normfinder, and Comparative delta CT [
35], to identify the best suitable circulating miRNAs in the plasma of tuberculosis-infected and non-infected controls. The computation using Normfinder algorithms [
21] revealed that all the five selected miRNAs had a stability value of less than 1, indicating that they can be selected as a reference marker. However, miR-22-3p was the most stable, with a value of 0.485 (
Table 4 and
Figure 3). Similarly, analysis of qPCR data using comparative delta Ct methods [
23] also proved that miR-22 was the most stable endogenous, with a gene stability value of 0.949 (
Figure 5 and
Table 6). In line with our observations, miR-22 combined with miR-26a and miR-221 were also proposed as reference miRNAs for circulating miRNA in hepatitis B infected patients [
36].
Evaluation of miRNAs expression data performed using Bestkeepers modeling [
22] revealed that miR-93-5p, miR-22-3p, and miR-16-5p had acceptable standard deviations (i.e., <1). Of them, miR-93-5p was the most stable with a value of 0.82, followed by miR-22-3p and miR-16-5p with SDs of 0.90 and 0.94, respectively (
Table 5 and
Figure 4). In addition, the computation of qPCR data using geNorm [
24] resulted in both miR-93-5p and miR-22-3p as stable reference miRNAs with stability values of 0.629 (
Figure 6 and
Table 7), which is comparable to a cohort study conducted to normalize the expression of circulating miRNA in the plasma of tuberculosis-infected and non-infected participants, which reported that miR-93 was the most stable reference gene [
17].
It has been well-reviewed that miR-93-5p is one of the most common circulating miRNAs reported as an internal reference control for both cancer and other diseases [
15]. Song et al. employed multiple algorithm tools to analyze the stability of circulating microRNA, and reported miR-93, combined with miR-16, as a stable serum miRNA for gastric carcinoma patients and healthy controls [
37]. In another study, miR-93-5p, together with miR-25-3p and hsa-miR-106b-5p, were proposed as internal references for serum miRNA in colorectal cancer patients [
38]. Similarly, miR-93-5p and miR-425-5p were identified as stable endogenous markers in the plasma of vulvar carcinoma [
39] and miR-93 with miR-101-3p in the plasma of individuals associated with major depression disorder [
40].
Finally, comprehensive ranking analysis, which examined the overall geometric ranking [
35] showed miR-22-3p as the most stable, followed by miR-93-5p with values of 1.19 and 1.73, respectively (
Figure 7 and
Table 8), whereas miR-16-5p was the least stable, with a value of 2.83. In general, the ranking order of stability was as follows: miR-22-3p > miR-93-5p > let-7i-5p > let-7a-5p > miR-16-5p (
Table 9).
In summary, the stability and gene expression of circulating miRNA can be affected by various conditions associated with either the host or environment, or both. Thus, setting an optimal endogenous control is important to get a reliable result that indicates the clinical conditions. Our study implicated that miR-22-3p and miR-93-5p were stably expressed and can be utilized as an endogenous reference to normalize gene expression data for circulating miRNAs obtained from plasma of tuberculosis-infected and non-infected health controls.