**4. Preclinical study and animal trials**

Historically, most studies of revascularization have been based on and reported according to angiographic criteria. Some patients with significant arteriosclerosis of the heart are not amenable to revascularization of a coronary artery because they have a combination of microangiopathy and significant macroangiopathy. Therefore cardiac surgeons developed the technical approachof venous revascularization. Several systematic reviewshave beenconduct‐ edinanattempttodefine the exactrole of animalmodels asplatforms forfuturehumantherapy [9-12].We investigated the benefit of arterialization of a cardiac vein under these circumstan‐ ces in some animal models [13] which indicate retrograde venous revascularization is possi‐ ble and improves cardiac function in a state of acute ischemia so we could find its way into practical use in coronary heart surgery. In experimental studies in a variety of animals and in human clinical studies, retroperfusion of the coronary sinus has been used to improve myocar‐ dial perfusion and postischemic systolic and diastolic function in many surgical procedures. In addition, animal trials, mostly involving sheep, dogs and pigs showed that arterialization of cardiac veins decreases infarct size as well[11,14]. These animal models are likely to be useful for pre-clinical evaluation of the functional effects of surgical therapy.


**4. Conclusion**

This chapter has been focused on one-step physical synthesis of Ge/TiO2 and PbSe/ZnSe composite thin film as candidate materials for quantum dot solar cell. It should be pointed out in the Ge/TiO2 that the anatase-dominant structure appears in the restricted composition range as a result of optimization of Ge chip numbers and additional oxygen ratio in argon. Furthermore, their optical absorption edge is obviously shifted to vis-NIR region. The solubility range of Ge in the Ti1-*x*Ge*x*O2 powder is estimated to be 0.23 0.01 at 1273 K. In addition, their optical absorption edge is obviously shifted to the UV region as *x* increases. Thus, the Ti1-*x*Ge*x*O2 solid solution does not exhibit the vis-NIR absorption. In contrast, SAXS and HREM results clearly indicated that the Ge nanogranules were embedded in the matrix. The size was sufficiently small to appear the quantum size effect. Thus, the both valuable characteristics are simultaneously retained in the Ge/TiO2 composite films. In the PbSe/ZnSe, the solubility limit of Pb in ZnSe is quite narrow, less than 1mol% in the film form, indicating that an atmosphere near thermal equilibrium is achieved in the apparatus used. Elemental mapping indicates that isolated PbSe nanocrystals are dispersed in the ZnSe matrix. The optical absorption edge shifts toward the lower-photon-energy region as the PbSe content increases. In particular, onset absorption can be confirmed at approximately 1.0eV with 16mol%PbSe, favorably covering the desirable energy region for high conversion efficiency. The insolubility material system and the HWD technique enable a one-step synthesis of PbSe/ZnSe composite thin film.


**8. Opioids**

information, clinicians can refer to neuropathic pain guidelines that provide evidence‐based recommendations for specific disease state‐induced neuropathic pain [29]. If acute or chronic pain is completely neuropathic in nature, the WHO's stepwise ladder is not appropriate to

After the addition of nonopioids and possible adjuvants, the WHO's analgesic ladder calls for low to strong opioids. However, if a patient is in severe acute pain, it is reasonable to start therapy with all three types of analgesics and then rapidly de‐escalate to a lower step on the analgesic ladder [9]. When treating chronic pain, opioids should never be utilized as first‐line agents outside of cancer or palliative care. Opioids have only been shown in the literature to have a short‐term improvement in pain and carry a high risk for serious side effects and possibly even death [6]. Before initiating therapy, clinicians and patients should have a dis‐ cussion regarding expected goals and potential risks. Goals should include how efficacy will be measured for both pain relief and functionality and what measures will indicate contin‐ ued treatment. Patients should also be informed that opioids only show a short‐term benefit in relieving pain and long‐term efficacy is lacking. Expectations should be that opioids will

The addition of opioids to a chronic pain regimen should be considered carefully. Patients do not need to fail nonopioids or adjuvants prior to initiating opioids, benefits must simply out‐ weigh the risks of starting opioid therapy [1, 8]. Another way to consider this is that opioids should be considered in patients with severe disabling pain that is likely not to be relieved from nonopioids and adjuvants alone. The worst risks are with overdose and potentially fatal respiratory depression. Overdose risk is dose dependent, and clinicians should be care‐ ful when doses are escalated. This is particularly important as there is technically no ceiling dose for opioids. Several other factors increase the risk for opioid‐related overdose including methadone use, co‐prescription with benzodiazepines, history of sleep‐disordered breathing, reduced renal or hepatic function, increased age, pregnancy, history of substance abuse and psychiatric illness. Additionally, the risk of opioid‐related overdose is elevated when starting patients on opioid therapy with long‐acting or extended release formulations. For this reason, these dosage forms should only be utilized in opioid‐tolerant patients. Risk mitigation strate‐ gies such as checking prescription refill history, urine drug screening and use of medications specifically for opioid use disorders (methadone, buprenorphine) increase retention in opioid

Prior to starting opioid therapy, the prescriber must fully understand the concepts of opioid abuse (opioid misuse disorders), tolerance and physical withdrawal [6]. Opioid abuse or opioid misuse disorders are described by patterned misuse of opioids that include unsuccessful attempts to curb use and results in social problems at home, work or school. Tolerance is simply a diminished response to a fixed dose of medication with repeated use. Physical dependence is when a medication causes the body to change in a way that when the medication is removed the body produces withdrawal symptoms. Both

follow and a medication regimen targeting neuropathy should be initiated.

**7. Risk of opioids**

194 Pain Relief - From Analgesics to Alternative Therapies

likely never provide complete relief.

treatment programs.

It is believed that opium was cultivated in Mesopotamia as early as 3400 BC [35]. Natural occur‐ ring opiates are the alkaloid compounds found in the poppy plant and include morphine and codeine while the term opioid refers to any compound that binds to opioid receptors. Narcotic originally was used to describe a medication that causes sleep, but the common misuse of drugs like quaaludes and barbiturates along with opioids caused this to become an umbrella term for drugs that are commonly abused. It is even used in a legal sense to describe the drugs of abuse. Even though opioids are grouped together, they have a wide range effects and each medication has unique properties (**Table 2**). There are four major opioid classes, and understanding each of the groups allows for easier prescribing as efficacy and side effects are similar within classes.

**Table 2.** Properties of common opioids.

originally was used to describe a medication that causes sleep, but the common misuse of drugs like quaaludes and barbiturates along with opioids caused this to become an umbrella term for drugs that are commonly abused. It is even used in a legal sense to describe the drugs of abuse. Even though opioids are grouped together, they have a wide range effects and each medication has unique properties (**Table 2**). There are four major opioid classes, and understanding each of the groups allows for easier prescribing as efficacy and side effects are similar within classes.

> **Starting oral dose (mg)**

30 30–60 Immediate release:

7.5 4–8 Immediate release:

10 5–10 Immediate release:

20 15–30 Immediate release:

– 0.025 Transdermal: 24

4–6

8–24

200 30–60 4–6 Only available in

4–6

12–24

30 5–7.5 4–6 Only available in

4–6

12

4–6

12

Extended release:

Extended release:

Extended release:

Submucosal:

Extended‐release:

**Dosing interval (h) Comments**

Several different extended‐release formulations each with their own dosing interval recommendations Strong opioid

combination with paracetamol Weak opioid

Several different extended release formulations each with their own dosing interval recommendations Strong opioid

combination with paracetamol Weak opioid

Strong opioid

Can use lower starting doses if using combination product with paracetamol Weak opioid (combination product) Strong opioid (when used alone at higher doses)

Use only in patients suffering from severe chronic pain

**Equianalgesic oral dose (mg)**

**Drug name pertinent dosage forms**

> release extended release (Ms contin and kadian)

196 Pain Relief - From Analgesics to Alternative Therapies

release (combination with paracetamol)

release Extended release

release (combination with paracetamol)

release Extended release

release (alone and combination with paracetamol) Extended release

Submucosal

Morphine Immediate

Codeine Immediate

Hydromorphone Immediate

Hydrocodone Immediate

Oxymorphone Immediate

Oxycodone Immediate

Fentanyl Transdermal;

The phenanthrenes are one of the larger classes of opioids and contain the prototypical opioid morphine. This class contains the most commonly used opioids including morphine, codeine, hydrocodone, hydromorphone, oxycodone, oxymorphone, levorphanol, buprenorphine, nal‐ buphine and butorphanol [35]. A majority of these agents are metabolized in the liver by the CYP450 isoenzyme CYP2D6. Some even require metabolism to exert analgesic effects. For example, codeine itself has no analgesic effect in its original state, and it is only through metabolism by CYPD6 to morphine that it can produce an analgesic effect [36]. A similar process happens to both hydrocodone and oxycodone to be converted to hydromorphone and oxymorphone. Similar to codeine, hydrocodone has been proposed to be a prodrug and its analgesic effect is dependent on activation by CYP2D6. Oxycodone, on the other hand, is a μ‐opioid agonist and does not require activation by CYP2D6. A serious issue arises with the fact that CYP2D6 has a very dramatic range of activity from one person to the next. It has been reported in literature that through a mutation, some patients have no activity of CYP2D6 (codeine produces no analgesic effect) whatsoever while others may be classified as ultrar‐ apid metabolizers. This may explain the wide range of reported efficacy in patients who are prescribed codeine‐ and hydrocodone‐containing products.

Another common attribute of drugs in the phenanthrene class is that they are typically gluc‐ uronidated and eliminated via the kidneys. This is especially important for morphine whose glucuronidated metabolite, morphine‐6‐gucuronide, is responsible for its analgesic effects [37]. In young and healthy individuals, this is not of importance, but in elderly or those with markedly reduced renal function, morphine's analgesic effects are prolonged. Morphine prescribed at fixed intervals in this patient population should be closely monitored as the respiratory side effects may accumulate as the medication is cleared more and more slowly. Additionally, drugs in this class with a 6‐hydroxyl group (morphine and codeine) are associ‐ ated with a higher incidence of nausea than those in the class that do not [35].

Phenlyperidines include the agents' fentanyl, alfentanil, sufentanil and meperidine. Of these, fentanyl has the highest affinity for the μ‐opioid receptor and is 80–100 times that of mor‐ phine [35, 37]. While incredibly potent fentanyl has a very short half‐life leading to a short duration of action. Fentanyl's only advantage is that is highly lipophilic leading to its ability to be utilized nontraditional dosage forms. One of these dosage forms is the transdermal patch. Fentanyl transdermal patches should only be used in the most extreme cases of chronic low back pain. This dosage form possesses many nuances, and a complete understanding of them should be obtained before prescribing. When a fentanyl transdermal system is placed on a patient, it takes 6–12 h before taking effect [37]. Additionally, it will take 3–6 days to reach steady state and when removed a reservoir of drug will remain in effect up to 24 h. This makes initiating and weaning incredibly difficult and therefore should not be commonly done. Fentanyl also has a submucosal dosage form that may be beneficial in patients who suf‐ fer from acute breakthrough pain. The clinically applicability of this makes sense because of fentanyl's high potency and short duration of action. It is important to stress that this should not be prescribed on a regular basis and should only be utilized as a rescue medication in very rare cases. The majority of patients with chronic low back pain should not be prescribed fentanyl, but in rare circumstances, it may have clinical utility.

The other opioids in the phenlyperidine class should not be used in the treatment of low back pain. Meperidine is a relatively weak opioid agonist with poor oral absorption that fell out of favor due to its neurotoxic and anticholinergic side effects [23, 37]. Its metabolite normeperidine accumulates in patients with renal insufficiency and lowers seizure threshold. Additionally, it has significant drug‐drug interactions with monoamine oxidase inhibitors that lead to severe respiratory depression. The other agents that are sufentanil and alfentanil in this drug class have little to no role in the treatment of low back pain due to their short duration and lack of specialized dosage forms.

The remaining classes of opioids consist of the benzomorphans and the diphenylheptanes. The only agent in the benzomorphan class is pentazocine, which is a mixed opioid agonist‐ antagonist. The diphenylheptaines include propoxyphene and methadone. While metha‐ done has established itself in a highly specific role, propoxyphene has fallen out of favor dramatically [16]. Propoxyphene is thought to be no more efficacious than paracetamol, and it has a plethora of side effects. It has been associated with dizziness, weakness, paradoxical excitement, falls, visual disturbances and insomnia [35]. Propoxyphene itself is thought to act directly on the central nervous system and increase the risk for seizure activity which ulti‐ mately leads to the product being pulled off the market in the United States.

Several opioids have mixed agonist‐antagonist activity and have only a limited role in the treatment of pain. These drugs provide small analgesic effects in patients with little or no prior opioid exposure and may exacerbate withdrawal symptoms in patients who have a physical dependence on opioid medications [35, 37]. Drugs in this group are pentazocine, butorphanol and nalbuphine. These agents have a ceiling effect on both analgesia and respiratory depres‐ sion and have limited abuse potential. As doses escalate so do the antagonistic effects against other opioids. This places the patient at risk for withdrawal. Also, the risk for psychotomi‐ metic side effects (delirium, delusions and hallucinations) increases in conjunction with the dose [37]. Pentazocine has the highest incidence of these side effects. The role of these agents in pain is limited by their antagonistic effects and lack of convenient dosage forms. Only pentazocine is available in an oral dosage form and has fallen out of favor to treat acute or chronic pain. The partial agonist buprenorphine acts similarly to the agonist‐antagonists with the one caveat that it is not associated with psychotomimetic side effects. Similar to the ago‐ nist‐antagonists, it can produce withdrawal symptoms when administered to patients taking high doses of other opioid medications. It is also combined with naloxone to reduce the risk of abuse. Buprenorphine is available as a sublingual dosage form in the United States and a transdermal extended release system in Europe. The sublingual form is commonly used in the United States in addiction treatment programs.

Phenlyperidines include the agents' fentanyl, alfentanil, sufentanil and meperidine. Of these, fentanyl has the highest affinity for the μ‐opioid receptor and is 80–100 times that of mor‐ phine [35, 37]. While incredibly potent fentanyl has a very short half‐life leading to a short duration of action. Fentanyl's only advantage is that is highly lipophilic leading to its ability to be utilized nontraditional dosage forms. One of these dosage forms is the transdermal patch. Fentanyl transdermal patches should only be used in the most extreme cases of chronic low back pain. This dosage form possesses many nuances, and a complete understanding of them should be obtained before prescribing. When a fentanyl transdermal system is placed on a patient, it takes 6–12 h before taking effect [37]. Additionally, it will take 3–6 days to reach steady state and when removed a reservoir of drug will remain in effect up to 24 h. This makes initiating and weaning incredibly difficult and therefore should not be commonly done. Fentanyl also has a submucosal dosage form that may be beneficial in patients who suf‐ fer from acute breakthrough pain. The clinically applicability of this makes sense because of fentanyl's high potency and short duration of action. It is important to stress that this should not be prescribed on a regular basis and should only be utilized as a rescue medication in very rare cases. The majority of patients with chronic low back pain should not be prescribed

The other opioids in the phenlyperidine class should not be used in the treatment of low back pain. Meperidine is a relatively weak opioid agonist with poor oral absorption that fell out of favor due to its neurotoxic and anticholinergic side effects [23, 37]. Its metabolite normeperidine accumulates in patients with renal insufficiency and lowers seizure threshold. Additionally, it has significant drug‐drug interactions with monoamine oxidase inhibitors that lead to severe respiratory depression. The other agents that are sufentanil and alfentanil in this drug class have little to no role in the treatment of low back pain due to their short

The remaining classes of opioids consist of the benzomorphans and the diphenylheptanes. The only agent in the benzomorphan class is pentazocine, which is a mixed opioid agonist‐ antagonist. The diphenylheptaines include propoxyphene and methadone. While metha‐ done has established itself in a highly specific role, propoxyphene has fallen out of favor dramatically [16]. Propoxyphene is thought to be no more efficacious than paracetamol, and it has a plethora of side effects. It has been associated with dizziness, weakness, paradoxical excitement, falls, visual disturbances and insomnia [35]. Propoxyphene itself is thought to act directly on the central nervous system and increase the risk for seizure activity which ulti‐

Several opioids have mixed agonist‐antagonist activity and have only a limited role in the treatment of pain. These drugs provide small analgesic effects in patients with little or no prior opioid exposure and may exacerbate withdrawal symptoms in patients who have a physical dependence on opioid medications [35, 37]. Drugs in this group are pentazocine, butorphanol and nalbuphine. These agents have a ceiling effect on both analgesia and respiratory depres‐ sion and have limited abuse potential. As doses escalate so do the antagonistic effects against other opioids. This places the patient at risk for withdrawal. Also, the risk for psychotomi‐ metic side effects (delirium, delusions and hallucinations) increases in conjunction with the

mately leads to the product being pulled off the market in the United States.

fentanyl, but in rare circumstances, it may have clinical utility.

duration and lack of specialized dosage forms.

198 Pain Relief - From Analgesics to Alternative Therapies

Methadone is another opioid with a unique mechanism of action other than being a μ‐opioid receptor agonist. This mechanism may be particularly advantageous for patients who have "opioid‐resistant" pain states or have a neuropathic component to their pain [37]. Similar to some of the agents to treat neuropathy, the R‐isomer of methadone is antagonistic at the NMDA receptor and may be beneficial in treating the effects of hyperalgesia and allodynia seen in chronic pain states [37]. Methadone should be used with caution as mentioned before its use increases the risk for overdose and a misunderstanding of its pharmacokinetics and pharmacodynamics perpetuates this effect. The terminal half‐life of the drug is typically thought to be 15–60 h, but has been cited as up to 120 h [38]. Because of this, it may take a week or longer to reach steady state, and therefore, the drug should be titrated no more often than weekly. Additionally, the analgesic effect of methadone is roughly 4–8 h and should be dosed on an every 8‐h interval. Due to this discrepancy, the drug has a high risk for accumu‐ lation and may put the patient at risk for sedation, confusion, respiratory depression, cardiac abnormalities and death. The general consensus is that a dose of 2.5 mg every 8 h is a safe starting dose for opioid‐naive patients [38]. Careful monitoring should be performed on any patient starting on methadone. Another caveat to dosing methadone is that it has a nonlinear equianalgesic conversion. This means that patients on higher doses of opioids are more sen‐ sitive to the effects of methadone and when converting the ratio of morphine equivalents to methadone dose decreases. However, with caution, the practitioner can utilize this effect to their advantage in treating the most complicated of patients.

Lastly, there are two agents that are sometimes considered opioid analgesics, but have both opioid and nonopioid mechanisms. These two agents are tramadol and tapentadol. While both have activity at the μ‐opioid receptor, this activity alone does not equate to the full analgesic effects seen with these agents [35, 39]. The remainder of their analgesic effects can be attributed to the inhibition of serotonin and norepinephrine reuptake. Similar to TCAs, this may be useful in treating neuropathic pain and caution should be used when combining therapy with other antidepressants or medications that increase the levels of serotonin as it increases the risk for serotonin syndrome [29]. Tramadol can be used for mild‐to‐moderate pain, but it should not be used as monotherapy when opioids are indicated based on the severity of pain. Tramadol's analgesic effect is at most equal to codeine and is probably less than that of hydrocodone [13, 16]. The maximum dose of tramadol is 100 mg every 6 h. Higher doses than 400 mg a day should not be used as it increases the risk for lethargy, nausea, tachy‐ cardia, agitation and hypertension. Additionally, tramadol has a neuroexcitatory effect so as doses increase so does the risk for seizures [13]. For these reasons, tramadol should be used either as an adjuvant medication or prior to stepping up to moderate or strong opioid‐con‐ taining regimens and is often seen as adjunct medications.


*Review* **Considerations and Suggestions for the Reliable Analysis of miRNA in Plasma Using qRT-PCR**

**Eunmi Ban and Eun Joo Song \***

Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 03760, Korea<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** MicroRNAs (miRNAs) are promising molecules that can regulate gene expression, and their expression level and type have been associated with early diagnosis, targeted therapy, and prognosis of various diseases. Therefore, analysis of miRNA in the plasma or serum is useful for the discovery of biomarkers and the diagnosis of implicated diseases to achieve potentially unprecedented progress in early treatment. Numerous methods to improve sensitivity have recently been proposed and confirmed to be valuable in miRNA detection. Specifically, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is an effective and common method for sensitive and specific analysis of miRNA from biological fluids, such as plasma or serum. Despite this, the application of qRT-PCR is limited, as it can be affected by various contaminants. Therefore, extraction studies have been frequently conducted to maximize the extracted miRNA amount while simultaneously minimizing contaminants. Moreover, studies have evaluated extraction efficiency and normalization of the extracted sample. However, variability in results among laboratories still exists. In this review, we aimed to summarize the factors influencing the qualification and quantification of miRNAs in the plasma using qRT-PCR. Factors influencing reliable analysis of miRNA using qRT-PCR are described in detail. Additionally, we aimed to describe the importance of evaluating extraction and normalization for reliable miRNA analysis and to explore how miRNA detection accuracy, especially from plasma, can be improved.

**Keywords:** qRT-PCR; plasma; miRNA; amplification efficiency

### **1. Introduction**

Circulating microRNAs (miRNAs) are highly stable extracellular molecules that circulate in the bloodstream [1,2]. These circulating miRNAs are approximately 22 nucleotides in length and play an important role in gene regulation by binding to and repressing the activity of specific target messenger RNAs (mRNAs). Profiles of miRNAs in plasma and serum have been found to be altered in cancer and other disease states [3,4]. Numerous studies have reported that specific miRNA expression profiles are associated with pathological conditions such as cardiovascular disease [5], cancer [6] and other diseases [7], which may provide diagnostic and therapeutic value as biomarkers. In previous studies, elevated plasma expression levels of miRNA-499 [8], miRNA-122 [9] and miRNA-155 [10] are known to be associated with AMI, liver injury, and inflammation, respectively. Meanwhile, the plasma expression levels of miRNA-34 [11] and miRNA-23a [12] are known to decrease in solid tumors and lung cancer, respectively.

Therefore, analyses of circulating miRNAs are important for the discovery and study of disease biomarkers that may aid in disease risk assessment, diagnosis, prognosis, and monitoring of treatment responses (Figure 1).

Currently, miRNA levels in biological fluids, tissues, and cells are measured after extraction by commercial RNA extraction kits, such as chloroform–phenol-based extraction [13,14], magnetic bead extraction [15], and column-based extraction [16], followed by microarray [17,18], Northern blotting [19,20], and quantitative reverse-transcription

the Reliable Analysis of miRNA in Plasma Using qRT-PCR. *Genes* **2022**, *13*, 328. https://doi.org/10.3390/

**Citation:** Ban, E.; Song, E.J. Considerations and Suggestions for

Academic Editors: Giuseppe Iacomino and Fabio Lauria

genes13020328

Received: 5 January 2022 Accepted: 8 February 2022 Published: 10 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

polymerase chain reaction (qRT-PCR) analysis [21,22]. Among these methods, qRT-PCR is widely preferred over other detection methods because of its high sensitivity and specificity for detecting low levels of circulating miRNAs in plasma and serum (Figure 2).

**Figure 1.** The number of PubMed search results regarding articles reporting on analyses of circulating miRNAs.

**Figure 2.** Methods for endogenous miRNA analysis in plasma or serum compared in terms of the proportions of articles reporting their use among PubMed search results from the past 10 years.

However, in previous studies, plasma miRNA levels have varied according to the laboratories performing the measurements [23,24]. This can mainly be due to differences in sample processing, measurements and data analysis [25–28]. In general, accurate miRNA measurement using qRT-PCR requires a high-quality sample, especially because of the low concentration of miRNA in plasma. Therefore, to increase the extraction efficiency and consistency of miRNAs analysis obtained from plasma and serum, numerous efforts over several years have focused on various highly sensitive and specific methods of miRNA extraction from plasma [29–31]. Additionally, similar efforts targeting data normalization [32,33] and the optimization and assessment of PCR conditions have been pursued to improve the accuracy of PCR measurements [34,35]. However, few reviews have focused on the importance and necessity of evaluating PCR conditions for optimizing extraction conditions.

In this review, which focuses on quantity and quality, we describe factors influencing miRNA measurement in plasma and serum using qRT-PCR. Additionally, we review the advantages of assessing PCR efficiency and normalization to obtain reliable and accurate PCR-based results of miRNA analysis in plasma. This evaluation calls attention to the importance of the assessment of PCR efficiency for optimizing PCR and miRNA extraction conditions.

### **2. Inconsistent Measurement of miRNA Extracted in Plasma Using qRT-PCR**

Since miRNA analysis using qRT-PCR greatly depends on the quality of the miRNA extract, the results of miRNA analysis are different depending on the sampling procedure applied to the same sample. Many researchers have reported this inconsistency in miRNA analysis results, and various efforts are being made to analyze the factors that cause analysis inconsistency during the sample extraction step [31,36–38]. Brunet-Vega et al. compared the Cq levels of miRNAs extracted from the same plasma samples using five commercially available miRNA extraction kits [37]. They reported that the levels of the tested miRNAs were similar, but the levels of the spiked-in exogenous miRNAs were different. For this reason, the analyzed endogenous miRNAs were measured differently from the spike-in exogenous miRNAs. Another research group presented differences in the recovery and levels of miRNAs tested using two different miRNA extraction processes [31]. They also showed that the levels of tested miRNAs were affected by various sample treatments during the sample extraction process. Poel et al. also presented the effect of different carriers and pretreatment times on miRNA extraction recovery and showed inconsistent results between studies [38]. These studies recommended the need for standardization of protocols, including sample handling and extraction processes, to reduce the mismatch results of miRNAs in plasma between laboratories and between assays to perform reliable biomarker screening and discovery of miRNAs in plasma samples.

### **3. Factors Inhibiting Accurate miRNA Measurement in Plasma Using qRT-PCR**

miRNA levels in plasma are low—10- and 100-times less than the concentrations in cells and tissues, respectively [39]. Therefore, the reliable and accurate analysis of miRNAs in plasma is a major issue, despite significant developments in the field. Among various analytical methods, qRT-PCR is usually used to analyze circulating miRNA levels in biological fluids, including plasma and serum, owing to its high specificity. However, qRT-PCR analysis can be compromised by various materials, such as matrix and extraction residual reagents in samples, and consequently, miRNA analysis results can vary depending on the purity of the extracted sample. Specifically, the effect of the interference on miRNA analysis in plasma is larger in cells and tissues because of the low abundance of miRNAs in plasma. Therefore, many studies have investigated the interference of miRNA analysis in plasma using qRT-PCR, which is primarily caused by sample components and the residual reagents extracted [36,40].

### *3.1. Sample Matrix*

It is established that interference of qRT-PCR analysis is caused by various components present within the sample matrix. Therefore, for reliable qRT-PCR analysis, a high-purity sample from which components, such as proteins, lipids, and carbohydrates have been removed, must be prepared. The abundance of these confounding components varies significantly according to dietary status, anticoagulant type, as well as sampling and storage conditions [41,42]. Besides matrix components, hemolysis can be a major cause of variation in miRNA levels. Several miRNAs are found in large amounts in red blood cells (RBCs), and they are released from RBCs as a result of hemolysis, thereby increasing the level of certain miRNAs in the blood. However, hemolysis is more difficult to control than other conditions because it occurs frequently during blood sampling. Many authors have reported that miRNA qRT-PCR analysis results differ according to the degree of hemolysis of the sample. Myklebust et al. showed that qRT-PCR miRNA measurement is influenced by hemolysis [43]; in their study, the plasma miRNA concentration increased as the hemolyzed proportion of the sample increased, but the degree of increase depended on the miRNA type. Specifically, miR-16 is one of the most abundant miRNAs in RBCs [44], and many studies have shown that hemolysis may increase miR-16 levels in plasma. This is especially important because, due to its high abundance relative to other miRNAs, miRNA-16 is used as an endogenous reference gene to normalize the data after qRT-PCR analysis. Thus, hemolysis must be taken into consideration for accurate screening of blood miRNA levels. Feng et al. also demonstrated the effect of the sample matrix on miRNA analysis using qRT-PCR [36]. They showed variability in miRNA levels among matrices with varying compositions, including in terms of types or levels of anticoagulant molecules, plasma protein, and lipids, and hemolysis was analyzed using qRT-PCR. The authors showed that the factors influencing the sample matrix (leading to variability in miRNA analysis in plasma using qRT-PCR) are primarily associated with dietary status, anticoagulant selection, and plasma sample storage conditions. Mompeón et al. also demonstrated the effect of hemolysis on miRNA analysis using qRT-PCR, observing that miRNA levels differed between serum and plasma [45]. In addition to differences in sample components and conditions, there may be differences in the degree of interference with qRT-PCR analysis due to extraction efficiency. Variations in analysis results may also be associated with the purity of the prepared sample, which depends on the sample extraction method. For this reason, various sample extraction kits and methods have been developed, and some of these have been reported to reduce intralaboratory and interlaboratory variability. Several research groups have presented and compared commercial RNA extraction kits, and studies related to the standardization of miRNA extraction methods from biological fluids have been conducted [46–48]. Column-based extraction kits often obtain high-quality miRNA extracts, and they are associated with lower extraction variation chloroform–phenol-based kits. However, chloroform–phenol-based kits, such as Trizol, are also known for favorable extraction recovery and costs.

### *3.2. Residual Reagents*

For reliable qRT-PCR analysis, it is necessary to minimize any interfering factors using an extraction approach. For this reason, different extraction methods have been developed and applied to measure miRNA in cells, plasma, serum, and tissues. Currently, commercial miRNA extraction kits are largely divided into chloroform–phenol-based reagent kits and column-based extraction kits. Both methods include phenol extraction, which is a long-established approach to extracting nucleotides from biological samples. However, with phenol extraction, residual solvents, including phenol, remain in the final purified RNA sample after extraction and interfere with qRT-PCR-based miRNA analysis as a contaminant [49]. Among this interference caused by extraction reagents, residual phenol not only interferes with PCR analysis, but it can also cause errors in the quantification of RNA extracted from plasma. Specifically, the interference by residual phenol on miRNA analysis in plasma or serum is severe (relative to interference in cells and tissues) because of the low abundance of miRNAs in plasma and serum. For this reason, many researchers have investigated the issue of residual phenol. Spectrometric overestimations caused by residual phenol from extracted RNA yields have frequently led to inaccurate and variable plasma miRNA measurements. This problem is exacerbated by the fact that the wavelengths of RNA and phenol are similar, and the level of RNA in plasma is low compared with residual phenol. Several companies have, therefore, developed nanodrop systems to measure residual phenol concentrations in extracted samples to prevent mismeasurement of the amount or concentration of extracted RNAs by spectrometry [50,51]. Additionally, researchers have developed new systems that are not based on absorbance but that use specific fluorescent dyes for small RNAs to reliably measure extracted miRNAs [52,53]. For circulating biomarker detection analysis, accuracy could be optimized via the use of equal volume inputs rather than the same amount of RNA [54]. In plasma miRNA analysis by qRT-PCR, instead of direct RNA measurements, extraction recovery and analyzed samples are evaluated and normalized using spiked exogenous miRNA.

### **4. Important Considerations for Reliable miRNA Analysis Using qRT-PCR**

Plasma miRNA analysis is an important area of biological and clinical research that is gaining increasing recognition. However, low plasma miRNA levels are associated with miRNA measurement errors and consequent inaccurate analyses [32]. These errors are mainly caused by interference in the sample matrix. Therefore, researchers have attempted to evaluate interference and develop normalization methods to minimize errors caused by such interference.

### *4.1. Amount of miRNA*

Given that highly purified samples are needed for successful qRT-PCR analysis, many researchers have concentrated on developing methods that emphasize extracting highquality RNA rather than high yields. Column-based extraction kits are often used for RNA extraction from various sample types. However, high quantities of RNA are also needed for reliable miRNA analysis, but it is challenging to efficiently extract RNAs from serum and plasma [55]. For this reason, chloroform–phenol-based extraction is still used to extract miRNAs from plasma, although several researchers have discussed the problems associated with chloroform–phenol-based extraction methods in PCR analysis. Various extraction kits have been compared and investigated [53]. In addition to extraction kits, various trials have been conducted, including comparisons between different modifications of extraction processes (such as the addition of carriers to trigger precipitation or the modification of incubation conditions) to increase extraction recovery without loss of quality. Some research teams have optimized carrier types, concentrations, and incubation conditions to maximize plasma miRNA extraction [56,57].

### *4.2. Normalization*

Similar to other analytical methods, qRT-PCR analysis is subject to variations or errors, including in association with elements such as sample handling and volume measurements. Specifically, qRT-PCR plasma miRNA analysis can be greatly influenced by nutritional status, anticoagulant type, and plasma storage conditions. Therefore, normalization is important to mitigate variations in qRT-PCR analysis. One of methods for normalization of miRNA analysis by qRT-PCR is global normalization, which uses the calculated mean of all miRNAs in a given sample as the normalizer. This method is highly recommended when dealing with large scale miRNA expression profiling studies where several hundreds of miRNAs are analyzed [58]. However, global normalization cannot be applied for small-scale studies. Another popular method is normalization through reference genes. U6 is a small nuclear RNA commonly used as an endogenous internal control to normalize miRNA expression levels in different biological samples, including plasma. However, there is evidence that U6 plasma levels vary under certain conditions [59]. Therefore, various studies have been conducted to identify more reliable reference genes for normalizing endogenous plasma miRNA levels [60,61]. However, evaluations of different reported reference genes have yielded inconsistent findings [33,62–64]. Consequently, efforts have led to the identification of an appropriate endogenous miRNA for normalization, accounting for differences in plasma according to various factors, including disease status, sex, and age. External references, such as cel-miR-39-1 for normalization, are also frequently used to correct for extraction recovery and measurement. Zhang et al. showed the accuracy of normalization by reference gene candidates using exogenous miRNA (spiked-in cel-miR-39) as a target (Figure 3).

**Figure 3.** Effects of normalization by different reference gene methods on the expression levels of miRNAs from plasma samples of stable coronary artery disease patients and healthy controls (*n* = 5 in each group). For each group, 2 µL of exogenous cel-miR-39 was spiked into 300 µL plasma. The levels of cel-miR-39 were assessed by qRT-PCR and were normalized to miR-16, miR-6090, miR-4516, miR-484, and RNU6 [62].

Such an approach can eliminate multiple deviations of the experimental process, yielding more robust results. However, the approach also makes the experimental procedure cumbersome, and clinical applications more inconvenient. For example, cel-miR-39 was spiked into serum immediately before RNA extraction, allowing for the control of technical variation. However, the cel-miR-39 recovery was variable, ranging from 1% to 56%, thereby demonstrating the inherent need to take technical variability into account when performing absolute quantification [65]. Additionally, extraction kit-dependent differences in isolation yields across exogenous cel-miRs were reported. Nevertheless, the use of an exogenous cel-miR for normalization and correction presents less variability than strategies based on the concentration of endogenous components, such as the frequently used miR-16-5p [66]. Therefore, the normalization or correction strategy and, to a lesser extent, postanalytical concerns strongly limit the clinical implementation of miRNAs. To date, researchers are yet to establish a robust method of miRNA quantification for qRT-PCR that is clinically easy to implement and universally accepted.

### *4.3. Amplification Efficiency*

In each cycle, qRT-PCR automatically detects the PCR amplification of a specific gene target. In PCR analysis, the number of target sequence molecules should double during each replication cycle, corresponding to 100% amplification efficiency. However, in practice, inappropriate reaction conditions and polymerase inhibition affect primer template annealing, resulting in decreased amplification efficiency and potentially leading to inaccurate conclusions. The assessment of factors affecting amplification efficiency provides information regarding inappropriate or suboptimal reaction conditions, as well as the presence of contaminants interfering with accurate qRT-PCR analysis. Therefore, qRT-PCR assays result in significant uncertainty due to variations in amplification and extraction efficiency [67,68]. For these reasons, several studies have investigated amplification efficiency to improve the accuracy and reliability of qRT-PCR analysis [23,37,69–71]. Brunet-Vega et al. demonstrated the necessity of exogenous genes through circulating miRNA profiling analysis using a commercial RNA extraction kit and exogenous genes to control for technical factors affecting final miRNA levels. Additionally, the observation that PCR efficiency reduces the variability of miRNAs circulating between samples should be validated because miRNA analysis in plasma using PCR can be affected by samples and PCR components [37]. Lebuhn et al. also demonstrated interlaboratory variation in qRT-PCR miRNA analysis in terms of amplification efficiency according to qRT-PCR-related factors, including interlaboratory differences in extraction steps [23]. Zununi Vahed and our team have successfully optimized extraction conditions through evaluations of amplification efficiency for reliable endogenous miRNA analysis using qRT-PCR [69,70]. Table 1 shows extraction method-dependent differences in amplification efficiency and quantification cycle (Ct) values of extracted miRNA.

**Table 1.** Mean quantification cycle (Ct), PCR efficiency, and correlation–coefficient (*R 2* ) values of miR-21 isolation from cell lines, urine, and plasma by different methods.

Data from three biological replicates of cell lines (HT-29 and HUVEC), body fluids (plasma), and urine samples. (Reproduced from [69]).

Svec et al. reported on factors associated with effective amplification efficiency [71]. Given that polymerase inhibition is caused by contaminants transferred from the RNA isolation process or sample matrix, for factors related to PCR reaction conditions, extraction conditions should be evaluated and optimized through assessments of amplification efficiency to reduce contaminants interfering with accurate qRT-PCR analysis. These studies have revealed that contaminants have a greater effect on qRT-PCR-based miRNA analysis from plasma than samples such as cells and tissues due to low levels of plasma miRNAs. Therefore, potential sources of interference in extracted plasma samples must be identified and corrected based on amplification efficiency before conducting qRT-PCR analysis. Reaction conditions such as annealing and primer conditions must first be evaluated and optimized in terms of amplification efficiency to ensure accurate analysis. Importantly, based on the evaluation of amplification efficiency, the specificity and sensitivity of qRT-PCR results can differ by primer type and concentration [72].

### **5. Discussion**

Clinical and pharmaceutical research about plasma or serum miRNAs is becoming increasingly important. Consequently, endogenous plasma miRNA analysis has also become critical. Analysis of endogenous plasma miRNA is conducted using qRT-PCR, but such analyses have shown high variability between different laboratories and individuals [24]. One explanation for this is that miRNA levels in plasma are low, and qRT-PCR analysis is consequently affected by extraction and sample components. Therefore, several studies have investigated reproducible techniques and adjustments applied to miRNA analysis, such as sample extraction and normalization techniques. The high sensitivity of qRT-PCR as an analytical tool is matched by its sensitivity to interference by various factors. Therefore, the optimization of an effective extraction method is a major consideration for reliable PCR analysis, and many published articles report on extraction methods to minimize sample interference. Similarly, normalization and optimization of PCR conditions in terms of amplification efficiency have also been investigated, with consideration of issues, such as hemolysis, as major causes of interference. Specifically, normalization is heavily emphasized as an important factor facilitating reliable evaluation of plasma miRNA. Normally, reference genes are used to normalize endogenous miRNA, while exogenous

miRNAs, such as cel-39-1, can also be added to samples to compensate for differences in extraction efficiency between samples [37]. However, these do not reflect extraction recovery because extraction efficiency differs between endogenous and exogenous miRNA, as does the effect of amplification efficiency on the environment of the extracted sample. Therefore, in addition to exogenous miRNA, appropriate reference genes are needed to normalize extracted endogenous miRNA levels, and reference gene selection must be prioritized because some reference genes can differ depending on the sample condition and type. With normalization, the evaluation of amplification efficiency is also crucial for the reliability of qRT-PCR studies [37,73,74]. Sreedharan et al. demonstrated improvements in the reliability of expression data through primer-dependent improvements in amplification efficiency [73]. Variations in primer concentration and annealing temperature, as well as primer design, can affect amplification efficiency and consequently affect the reliability of expression data. Optimization of PCR and extraction conditions through assessments of amplification efficiency might be important determinants of accurate and reliable qRT-PCR analysis of endogenous plasma miRNA.

This review describes the considerable variation and poor reproducibility of qRT-PCRbased plasma miRNA analysis associated with incomplete optimization of extraction and RT-PCR conditions through amplification efficiency and normalization. In the context of evaluating amplification, the use of exogenous and endogenous reference genes for normalization is necessary for the reliable and reproducible quantification of circulating miRNAs in plasma. These factors should be considered when translating the analysis of circulating miRNAs from plasma and serum into validated biomarker-based tests for routine clinical use. However, despite advances, such as the standardization of extraction processes and normalization for reliable qRT-PCR analysis of plasma miRNA, issues remain regarding the accuracy of qRT-PCR analysis due to individual differences in matrix composition. Therefore, as shown in Figure 4, we propose that the optimization of extraction conditions and the evaluation and identification of dependable reference genes (based on assessments of amplification efficiency) are necessary to ensure reliable and robust qRT-PCR-based miRNA analysis necessity for future applications of circulating miRNAs.

**Figure 4.** Suggested flowchart for qRT-PCR analysis of plasma miRNA.

### **6. Conclusions**

This review presented factors influencing measurements of miRNAs in plasma/serum including assessment of PCR efficiency and normalization to obtain reliable and accurate

PCR-based results of miRNA analysis in plasma. It could suggest the necessity of the assessment of PCR efficiency for the optimization of PCR and miRNA extraction conditions. In this review, the effect of factors related to extraction efficiency such as sample matrix, residual solvent after extraction process and RNA amount was described among various factors influencing measurements of miRNAs using qRT-PCR. These factors may cause inhibitors of qRT-PCR analysis and consequently can lead to inaccurate qRT-PCR analysis. The necessity of amplification efficiency and normalization as another considerable part was reported for reliable and reproducible quantification of circulating miRNAs in plasma using qRT-PCR. From this review, we conclude that the optimization of extraction conditions and selection of reliable reference genes based on assessment of the amplification efficiency should be prioritized for achieving a reliable qRT-PCR-based miRNA analysis in plasma/serum.

**Author Contributions:** Conceptualization, E.B. and E.J.S.; investigation, E.B.; writing—original draft preparation, E.B.; writing—review and editing, E.B. and E.J.S.; supervision, E.J.S.; project administration, E.J.S.; funding acquisition, E.J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (2020R1A4A4079494 and 2019R1A2C2004052), as well as a Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (2021R1A6C101A442).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


3.7. Colorfulness

The colorfulness of a photo is in proportion to the number of nongrayscale pixels in the photo. An achromatic photo, on the other hand, is a grayscale photo. It is not necessary that less

Figure 11. Two photos with different degrees of balance: (a) more balanced; (b) less balanced.

Figure 12. Two photos with different degrees of colorfulness: (a) more colorful; (b) more achromatic.

Figure 13. Two photos with different degrees of simplicity: (a) simpler; (b) more complex.

colorful photos are low quality because professional photographers sometimes choose eliminating colors to express some feelings. As shown in Figure 12(a), the photo is colorful, while the photo in Figure 12(b) is achromatic, and they give different feelings. The degree of colorfulness of a photo is defined as the reciprocal of the achromatic feature that is a special color component feature because it comprises rare hue components and it is perceived as a grayscale photo. The achromatic feature can be obtained from

$$f\_{\text{achomatic}} = \sum\_{\mathbf{x}=1}^{\text{width}} \sum\_{y=1}^{\text{height}} \frac{|\{(\mathbf{x}, \mathbf{y}) | \mathbf{Ch}\_{\text{R}}(\mathbf{x}, \mathbf{y}) = \mathbf{Ch}\_{\text{G}}(\mathbf{x}, \mathbf{y}) = \mathbf{Ch}\_{\text{B}}(\mathbf{x}, \mathbf{y})\rangle|}{\text{width} \times \text{height}} \tag{10}$$

Then the degree of colorfulness yields

$$f\_{\text{colorfulness}} = 1/f\_{\text{achromatic}} \tag{11}$$

#### 3.8. Simplicity

Professional photos are usually possessed of greater simplicity to make the subject appear more attractive. Figure 13(a) is simpler in terms of its color distribution whereas the color distribution of Figure 13(b) is rather complex.

The simplicity feature is computed from the color distribution of a photo. The formula for the simplicity feature [6] is expressed as

On the Design of a Photo Beauty Measurement Mechanism Based on Image Composition and Machine Learning http://dx.doi.org/10.5772/intechopen.69502 107

$$f\_{\text{simplify}} = \left(\frac{|\langle l|k(\boldsymbol{\alpha}) \ni \gamma k\_{\text{max}}\rangle|}{4,096}\right) \times 100\% \tag{12}$$

where k(cl) is the color count for color cl, kmax is the maximum color count, and γ is set to 0.001. In this formula, the number of colors in the photo is reduced to 4,096; that is, the numbers of colors for R, G, and B are all reduced to 16, each of which is represented by 4 bits individually.


*3.3. Type/Scenario Evaluation*

Among the reviewed articles, there were 58 articles that addressed GSI type or scenario, 23 studies of which are directly based on local conditions and existing research results of GSI facility types for selecting one or more type(s) of GSI, while the other 35 articles are based on a number of preliminary alternatives from which the best solution is selected by evaluating multiple criteria. We do not deny the efforts of the former in terms of research caliber but endorse the reliability of the latter for reducing the weight of subjective judgments in decision making. When considering the infiltration function, for example, BR, IT, and PP may all be appropriate choices, if a solution is directly determined by a planner or institution; thus, strong subjective factors may be inevitable. The type/scenario evaluation process incorporates criteria from multiple dimensions of society, economy, and environment comprehensively, which makes the corresponding GSI planning more likely to be adopted and implemented. In addition, the evaluation of multiple alternatives is also in line with the trend in GSI planning of choosing combined facilities, as it can help to distinguish the functional differences between diverse facility types themselves and their combinations, and to broaden the research field of GSI. Hua et al. [62] simulated the two-dimensional runoff routing processes under different GSI scenarios and storm patterns via a hydrological model; they used an evaluation system consisting of life cycle cost analysis, analytic hierarchy process, and regret decision theory, the criteria of which covered technology, economy, environment, and operational aspects; lastly, the best strategy was determined as the combined use of bioretention, infiltration trenches, and rain barrels. Similarly, Kourtis et al. [63] proposed a framework for evaluating stormwater management measures in urban basins; they included hydrological, hydraulic, and economic criteria, aiming to quantify the impact of alternatives on mitigating urban flood, and evaluated the construction, operation, and maintenance costs of all scenarios based on a typical life cycle (30 years). Comparing the conventional scheme (offline detention tanks, sewer enlargement) and GSI (GR, PP) with no stormwater management measures, the results showed that the GSI solution performed more effectively when traffic congestion, noise, construction difficulty, and the impacts of coordination downstream are taken into comprehensive consideration. Accurate quantities of ES provided by GSI cannot be obtained currently; therefore, the values of evaluation criteria of water quality and quantity regulation services are not accurate enough. Consequently, the credibility of the best type/scenario through

this evaluation is still low, and therefore, it is urgent to quantitatively identify the values of ES provided by GSI.