Recovering circulating extracellular or cell-free RNA from bodily fluids

Georgios Tzimagiorgis a,*, Evangelia Z. Michailidou b, Aristidis Kritis c, Anastasios K. Markopoulos b,Sofia Kouidou a

a Laboratory of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, 540 06 Thessaloniki, Greeceb Department of Oral Medicine and Maxillofacial Pathology, Aristotle University, Thessaloniki, Greecec Laboratory of Physiology, Medical School, Aristotle University, Thessaloniki, Greece

1. Introduction

Despite the advances in cancer therapeutic approaches, duringthe last decades, the morbidity and mortality rates in cancer stillremain high [1]. The earliest possible diagnosis and treatment isstill the best approach to improve survival rates to the disease [2].The National Cancer Institute estimates that premature deaths,which may have been avoided through screening, range from 3% to35% [3]. Screening for cancer is usually attempted wheneverworrying symptoms arise, having as a result the diagnosis of canceras a late stage disease [4]. The current methods for diagnosis of thedisease are usually invasive (e.g. PAP smear, colonoscopy, etc.) andexpensive whereas the existing biological markers are notdefinitive and lack high sensitivity and specificity [5]. Manyresearchers therefore work on developing new, sensitive andinexpensive methods for cancer diagnostics [6]. Detection ofextracellular or cell-free nucleic acids (DNA or RNA) in plasma,serum and other bodily fluids using the PCR or RT-PCR methodshave been suggested as non invasive and cost effective methods forcancer detection [7]. The first report on nucleic acid detection inplasma, was back in 1972 [8] and since then numerous are the

reports on detection of cancer specific mutations in circulatingDNA or RNA in plasma [9], as well as in other bodily fluids [10–12].

More recently, mRNA biomarkers in serum, plasma and otherbodily fluids have been analyzed performing reverse transcription-PCR (RT-PCR)-based detection strategies in patients with cancer[13]. Parallel to the increasing number of such biomarkers in bodilyfluids is the growing availability of technologies that enable massscreening programs [14].

Detection of mRNA markers compared to conventionalbiochemical methods, seems to increase specificity, sensitivityand timeliness of the cancer screening method.

2. Origin of circulating extracellular or cell-free nucleic acids

Extracellular or cell-free nucleic acids (DNA or mRNA) havebeen isolated from various bodily fluids and used as biologicalmarkers for various diseases among which is cancer [15–18].Several terms are used for these extracellular nucleic acids such as:(1) Circulating nucleic acids, mainly used for DNA and RNA,circulating in plasma or serum (2) Extracellular, or (3) Cell-freenucleic acids, when isolated from other body fluids such as: saliva,urine, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid(BALF) amniotic fluid and other.

Several different mechanisms have been implicated as apotential source for the circulating extracellular or cell-freenucleic acids. One possible mechanism is cell necrosis resulting

Cancer Epidemiology 35 (2011) 580–589

A R T I C L E I N F O

Article history:

Received 16 April 2010

Received in revised form 28 February 2011

Accepted 28 February 2011

Available online 22 April 2011

Keywords:

Extracellular or cell-free RNA

Cancer detection

Biological fluids

RNA stabilizing reagents

RNA isolation

mRNA

miRNA

A B S T R A C T

The presence of extracellular circulating or cell-free RNA in biological fluids is becoming a promising

diagnostic tool for non invasive and cost effective cancer detection. Extracellular RNA or miRNA as

biological marker could be used either for the early detection and diagnosis of the disease or as a marker

of recurrence patterns and surveillance. In this review article, we refer to the origin of the circulating

extracellular RNA, we summarise the data on the biological fluids (serum/plasma, saliva, urine,

cerebrospinal fluid and bronchial lavage fluid) of patients suffering from various types of malignancies

reported to contain a substantial amount of circulating extracellular (or cell-free) RNAs and we discuss

the appropriate reagents and methodologies needed to be employed in order to obtain RNA material of

high quality and integrity for the majority of the experimental methods used in RNA expression analysis.

Furthermore, we discuss the advantages and disadvantages of the RT-PCR or microarray methodology

which are the methods more often employed in procedures of extracellular RNA analysis.

� 2011 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +30 2310999122; fax: +30 2310999004.

E-mail address: tzimagio@med.auth.gr (G. Tzimagiorgis).

Contents lists available at ScienceDirect

Cancer EpidemiologyThe International Journal of Cancer Epidemiology, Detection, and Prevention

jou r nal h o mep age: w ww.c an cer ep idem io log y.n et

1877-7821/$ – see front matter � 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.canep.2011.02.016

in the presence of high amounts of DNA (and probably RNA) inthe plasma of cancer patients with large or advanced tumors[19,20]. Apoptosis has also been considered as another possiblesource of circulating cell-free nucleic acids. Circulating DNAanalyzed by electrophoresis often exhibits a typical ladder offragmentation similar to that shown by apoptotic cells [21–23].Apoptotic bodies come from aged or damaged cells. Halicka et al.demonstrated that during apoptosis the nucleic acids, DNA andRNA, are packaged separately into two types of apoptotic bodies,one type contains only RNA and no detectable levels of DNA andthe other type contains only DNA and no detectable RNA [24].Furthermore, it was shown that extracellular, mRNA coding forthe enzyme tyrosinase which is associated with apoptoticbodies, is protected, from degradation in human serum [25].These finding suggests that extracellular RNA, circulating withinapoptotic bodies, is less susceptible to ribonucleases’ activities[5,25].

Spontaneous, active release of DNA and RNA by tumors inmicrovesicles is another possibility. Both normal but also cancercells release microvesicles which are spherical membrane frag-ments [26] and come from the surface of the cell or the endosomalmembranes. Microvesicles are released from viable cells and areusually smaller in size than the apoptotic bodies. Microvesicles arereported to be a normal constituent of human plasma [27]transferring proteins and RNA and contain no fragmented DNA[5,27–41]. In addition, lysis of cancer cells, trauma [5,42] andtherapeutic procedures [43–46] might also contribute to circulat-ing nucleic acids.

3. Biological fluids containing circulating extracellular (or cell-free) RNAs

3.1. Plasma/serum

The first reports on increased circulating nucleic acids – bothDNA and RNA – in the serum of cancer patients were back in 1977[47]. Circulating DNA and RNA in the serum of cancer patients wereisolated and characterized as tumor-derived and tumor-specific inthe late 1980s [23,19]. At first the cancer specific mRNA existing inserum was thought to come from circulating cancer cells [48]. Theamount of circulating nucleic acids however cannot be justified bythe number of cancer cells existing in serum [9], leading to thehypothesis that tumor specific circulating or cell-free nucleic acidscome from the necrosis [19,20] or apoptosis [24,25] of cancer cellsor they are even actively released by them in microvesicles[26,27,29].

Circulating RNA has been isolated from the plasma or serum ofpatients suffering from various types of malignancies such asbreast cancer [49–56], lung cancer [57–63], prostate cancer [64–78], thyroid cancer [79–98], hepatocellular carcinoma [5,72,75,99–112], melanoma [113–116], gastric cancer [117], renal cell cancer[118], oesophageal cancer [119,120], rectal carcinoma [121],gynecologic malignancies [122], pancreatic cancer [61], coloncancer [61], bladder cancer [16] and solid tumors [123], and hasbeen used as a biological marker either for the early detection anddiagnosis of the disease [64,100], or as a marker of recurrencepatterns [81,121], survival predictor [51], follow up and surveil-lance marker [79,82].

In particular, in breast cancer, measurement of serummetastasin mRNA has been proposed as a screening tool,predicting poor survival and distant metastases [51], whereasmeasurement of circulating 5T4 mRNA as having the potential toidentify patients who could benefit from a 5T4-targetedtherapy [53]. Moreover, the measurement of circulating Dkk-1 levels in breast cancer patients could predict bonemetastases [56].

In lung cancer, serum hTERT mRNA together with EGFR mRNAwas proposed as a good biomarker for diagnosis and clinical stageassessment [58].

In prostate cancer, serum PCA3 mRNA detection [64], HIF-1alpha mRNA quantitative RT-PCR [65], and serum E2F3 mRNAmeasurement [67], are proposed for the diagnosis of the disease.Furthermore, circulating PSA mRNA was proposed either as apreoperative prognostic marker for organ-confined or locallyadvanced prostate cancer [71], or as a tool to detect potentialsurgical failures pre-operatively [77] and monitor patients withmetastatic prostate cancer [69].

In thyroid cancer, peripheral thyrotropin hormone receptor(TSHR) mRNA has been proposed for cancer detection in patientswith thyroid nodules [87], or in indeterminate fine-needleaspiration [83] or for predicting recurrence [79]. Moreover someauthors suggest measurement of serum thyroglobulin mRNA indetecting thyroid cancer [87] or monitoring metastatic disease[89], whereas others do not support this idea [90,91].

In hepatocellular carcinoma, Dong et al. suggested thatmeasuring circulating plasma hepatic TGF-beta1 might proveuseful in the diagnosis and prognosis of HBV-induced hepatocel-lular carcinoma [100]. Wu et al. proposed hepatoma-specificalpha-fetoprotein mRNA (HS-AFP mRNA) and circulating alpha-fetoprotein (AFP)-mRNA [103] and Dong et al., insulin-like growthfactor II (IGF-II) mRNA [107] as markers to diagnose hepatocellularcarcinoma, monitor metastasis and relapse.

In esophageal squamous cell carcinoma, preoperative squa-mous cell carcinoma–antigen messenger RNA (SCC–Ag mRNA)levels in the peripheral blood are proposed as predictive factor forrecurrence [120].

The detection of circulating fetal RNA (placenta derived) in thematernal circulation has been tested in prenatal diagnostics as anon-invasive procedure compared to amniocentesis, chorionicvillus sampling and cordocentesis which are for the moment theroutine methods. The first to show that fetal RNA is present in theplasma of pregnant women was Poon in 2000 [124]. Ng et al. in2003 [125] showed that placenta-derived RNA coding for humanplacental lactogen and the b-subunit of human chorionicgonadotrophin and corticotrophin-releasing hormone (CRH)[126] are detectable using Real-time PCR in the maternal plasma.Detection of placental circulating RNA in the maternal plasma hasalso been proposed for the early diagnosis of several pregnancycomplications such as preeclampsia [127] and chromosomalaneuploidies [128]. These markers have been shown to beindependent of gender and polymorphisms [129]. Moreover,Oudejans et al. in 2003 [130], detected placenta-derived mRNAspecific for chromosome 21, whereas Wataganara et al. in 2004[131] detected fetal-derived gamma-globin mRNA in the maternalplasma. Further more in 2004, Tsui et al. [132] using expressionmicroarrays identified new mRNA species detectable in maternalplasma that can be used in the future as markers in prenataldiagnostics. Establishing these markers in the clinical diagnosticroutine is the next step to be taken.

Cell-free DNA was demonstrated to increase in the circulationof patients after acute trauma giving its measurement a possibleprognostic ability [133]. On this basis, there was an attempt tomeasure glyceraldehyde-3-phosphate dehydrogenase mRNA inthe plasma of trauma patients which was found significantlyincreased to that of healthy individuals when plasma was filteredthrough a 0.22 mm pore size filter [134]. Orlandi et al. attempted tomeasure circulating mRNA for nephrin in healthy volunteers andrenal transplantation patients [135]. They found that mRNA fornephrin reduces significantly with age and transplantationprobably due to the reduced renal mass. Circulating mRNA fornephrin was found however higher in females something whichmust be taken into account when accessing the results of cell-free

G. Tzimagiorgis et al. / Cancer Epidemiology 35 (2011) 580–589 581

mRNA for nephrin in plasma. Circulating cell-free RNA was alsodetected in the plasma of patients suffering from diabeticretinopathy in diabetes mellitus [136]. Two are the majorcomplications in diabetes mellitus, retinopathy and neuropathy.Circulating rhodopsin mRNA [137,138] and neuron-specificenolase mRNA [139] were found to significantly follow theseverity of the retinopathy.

3.2. Saliva

Saliva is the bodily fluid secreted in the oral cavity as theproduct of a set of 3 major salivary glands (the parotid,submandibular, and sublingual gland) and the numerous minorsalivary glands lying beneath the oral mucosa. It contains variousproteins, microorganisms, blood cells, desquamated epithelialcells, cell free-DNA and RNA and other components [140], forexample a breast cancer marker protein, c-erb-B-2, is reported tobe found in stratified saliva fractions [141,142]. RNA in the salivamay come from: (1) the secretions of the salivary glands (by theacinar cells or by the circulation [143], (2) passive diffusion fromserum to saliva across cell membranes [141,142], (3) desquamatedoral epithelial cells, (4) apoptotic oral epithelial cells [5,25], (5)blood cells or directly as cell-free RNA from blood released in salivathrough the crevicular fluid or microwounds, (6) cell death ortrauma and (7) even active release from epithelial or cancerouscells [26,27]. For years however RNA was thought to quicklydegrade in saliva due to the numerous RNases present in the saliva[144,145]. It was therefore a surprise that RNA molecules werediscovered intact in the saliva [18,146–162]. Detection of salivaryRNA has been used extensively in Forensic Medicine for body fluididentification [146–153], for the early diagnosis of oral squamouscell carcinoma [155,156], as genomic biomarker for primarySjogren’s syndrome [157], as a biomarker for the identification ofsleep drive in flies and humans [158]. Despite the opposite reports[159] salivary RNA generally appears to show stability andconsistency and has the potential to be used as a biomarker fororal cancer and possibly other diseases [143,154,160–162].

3.3. Urine

Numerous are the reports on the detection of various mRNAs inurine [163] for the diagnosis or surveillance of prostate [164,165]and bladder cancer [16,176–195], since urine is the bodily fluiddraining these tumors. Muthukumar et al. proposed the measure-ment of messenger RNA for FOXP3 in the urine of renal-allograftrecipients as a non invasive method for predicting acute rejectionof renal transplants [196]. Lately however, two reports on mRNAmarkers in urine concerning colorectal cancer [197] and metastaticmelanoma [198] have been published and raise the question ofhow these cancer specific mRNAs were present in urine.

PCA 3 (prostate cancer antigen 3) is the main cancer specificmRNA marker reported to have been detected in urine and hasbeen suggested for the diagnosis [164–171] and active surveillance[172,173] of prostate cancer. Molecular urine tests measuring theprostate cancer antigen 3 (RNA detection) are even commerciallyavailable [174,175]. Detection of PSA mRNA [165], TMPRSS2-ERGfusion [170] transcripts and Golgi protein GOLM1 [173] in urine asbiomarkers for prostate cancer have been also sporadicallyreported.

The detection of survivin, a member of the inhibitor of apoptosisfamily [176–181], cytokeratin 20 [177,182–185] and humantelomerase (hTERT) [186–191] mRNAs in urine have been reportedand used for the diagnosis and prognosis [16,177,182,194] ofbladder cancer or as a detection, characterisation and recurrencemarker [175,176,178–181,183–185,187–193,195] for the samedisease. The use of mucin 7 [177], hTERT [192], ETS2/urokinase

plasminogen activator [193], CD4 [183] and UHRF1 [194] mRNAshas been also reported as molecular markers for the diagnosis ofbladder cancer.

3.4. Cerebrospinal fluid (CSF)

In a study by de Bont the mRNA levels of insulin-like growthfactor-binding proteins, IGFBP-2 and IGFBP-3 were found signifi-cantly increased in paediatric medulloblastoma patients and theIGFBP-5 and IGF-II mRNA levels in paediatric ependymomapatients [199].

3.5. Bronchial lavage fluid

Free mRNA in bronchial lavage fluid has been suggested as anew diagnostic tool in lung cancer detection by Schmidt et al. in astudy published in 2005 [60].

3.6. miRNA

All the above mentioned studies refer to total and exogenousRNA or mRNA. Lately however there are some reports in theliterature concerning miRNA (both endogenous and exogenous) inplasma, serum, saliva and urine [200] that can be used asbiomarkers for the detection, monitoring and prognosis of cancer[201–221] and other diseases [217,222–226] or for body fluididentification [227]. Fully processed miRNAs are small RNAmolecules, approximately 18–24 nucleotides in length[220,228], which bind to mRNA and seem to regulate itstranscription [216]. miRNA can be isolated using the samemethods which are mentioned later in this document for theisolation of mRNA.

3.7. RNA stabilising reagents

RNA in biological samples and especially cell-free RNA in bodilyfluids is extremely unstable, degrading quickly after the samplecollection [229]. Several RNases are present in water, buffers, handsmear and various bodily fluids (tears, saliva and perspiration),which quickly degrade RNA in biological samples, cell cultures andexperiments. Therefore, the use of a method for stabilizing mRNAis important in order to ensure the isolation of a high qualitymRNA, preserving the gene expression profile.

Various methods have been used for the stabilisation of mRNA.These in general stabilise mRNA and help with handling thesamples at room temperature as well as in long term storage at�80 8C (archiving).

The first RNase inhibitor was isolated from placenta back in1979 [230]. Since then, several types of RNase inhibitors areavailable from many companies enabling RNA purification,transcription, translation, RT-PCR reactions, cDNA synthesis, etc.The various RNase inhibitors offered vary in the temperatures andpH at which they are active, the mechanism of their action, thetypes of RNases they block such as RNase A, B, C, T1 and S1 nucleaseand the need for dithiothreitol (DDT) addition for activity. Usuallythese RNase inhibitors do not block Taq polymerase, reversetranscriptase, RNA polymerase and other enzymes important forthe experiments. Some inhibitors are to be used for RNA isolation(RNAlater by Ambion, Qiagen, Superase-in by Ambion) (Table 1),and others are most effective as prophylactics during enzymaticreactions (Ribonuclease Inhibitor (Recombinant) by Affymetrix,RNasin1 Plus RNase Inhibitor and Recombinant RNasin1 Ribonu-clease Inhibitor by Promega, RNasin, Electrophoresis by SBSGenetech, RNaseOUTTM Recombinant Ribonuclease Inhibitor).

The most widely used RNase inhibitors are the placentalribonuclease inhibitors (RIPs) (Ribonuclease Inhibitor (Human

G. Tzimagiorgis et al. / Cancer Epidemiology 35 (2011) 580–589582

Placenta) – Affymetrix/USB, Ribonuclease inhibitor from humanplacenta – AppliChem, Placenta Ribonuclease Inhibitor – BioChain,etc.), which however are effective only against RNase A typeenzymes (RNAse A, B, C, human placental RNase) and not RNases 1,T1, T2, H, U1, U2 or CL3.

The type of RNase inhibitor used is dependent on theapplication. For example, Wong discusses upon RNAlater as asuitable RNase inhibitor for saliva that ‘‘RNAlater increases cellularmembrane permeability and its high salt content (ammoniumsulfate) changes intracellular salt concentration, leading to leakageof cellular components, including genomic DNA. RNAlatercompared with a number of RNA stabilizers was found to be theleast suitable, even worse than without any stabilizer’’ [231].

4. Methods for RNA isolation and analysis

4.1. Guanidinium-based protocols

The most commonly used techniques for rapid purification ofRNA are the methods employed guanidinium–acid–phenol extrac-tions [232–234]. RNA lysis buffers containing the chaotropicagents, guanidinium thiocyanate or guanidinium HCl, lead to theisolation of a very high quality and integrity of RNA. These reagentsare among the strongest protein denaturants and immediatelyinactivate RNases in the lysate. In all cases, protein denaturation(including disruption of RNases) is enhanced by the addition of theb-mercaptoethanol (b-ME). b-ME acts as a reducing agentbreaking intramolecular protein bisulfide bonds. The partitioningof RNA, DNA and protein in the resulting lysate is accomplished byacid–phenol extraction The RNA is then precipitated withisopropanol. Although the addition of exogenous RNase inhibitorsto eliminate both exogenous and endogenous nucleases is notrequired, the use of an RNase inhibitor in the RNA solutionenhances the stability of the stored material, and is highlyrecommended (see above).

4.2. Silica-columns based protocols

Recently, the development of new techniques employing silicacolumns for the isolation of nucleic acids provided new tools in theisolation of high quality extracellular RNA (Qiagen, Manual of viralRNA mini kit, Qiagen) [235]. This method is based on the formationof complexes between nucleic acids and the detergent (silica gelmatrix) in the presence of chaotropic salts. These complexes can beprecipitated by centrifugation to form a pellet. The resuspendedpellet, containing the desired RNA, is then treated with proteinaseK solution. The silica-based methods have several benefits, are lesslaborious and time-consuming, need less manipulation, involve nopellet handling, and therefore result to a reproducible high RNAyield [236]. All these advantages make the silica methods moreapplicable for routine molecular diagnostic methodology. In allcases, an internal control RNA can be added in the beginning of anyRNA isolation procedure in order to evaluate the method itself andmake a decision on which method is more suitable in yourresearch.

4.3. Isolation of DNA-free RNA from the samples

In all RNA experiments and particular in reverse transcription-polymerase chain reaction) (RT-PCR), it is crucial to eliminate anyresidual amount of DNA. Due to the sensitivity of PCR reaction, thepresence of genomic DNA contamination in an RNA preparationmay lead to ambiguous or misleading results. This problem isexaggerated when the analysis is performed using extracellularRNA, due to the small amounts of RNA obtained. To avoid thisproblem, it is highly recommended that RNA preparations betreated with DNase I (RNase-free) prior to cDNA synthesis[236,237], and that control reactions be performed in whichreverse transcriptase is omitted [238]. Furthermore, it is alsorecommended that all PCR primer pairs should be designed in amanner that the sequence for each primer may be located inadjacent exons of the gene of interest. This will allow thedistinction on the basis of size of the PCR products resultingeither from the amplification of the cDNA or from contaminatinggenomic DNA if there relatively small differences in the size [239].

4.4. Evaluation of the RNA quality obtained from the different samples

Although, there are a number of methods employed to obtainhigh quality RNA from a variety of sources, the isolation of highquality and integrity, extracellular RNA, is a very difficult task.Sample handling and variations in RNA isolation procedures mightaffect quality of the isolated and therefore could introduce errorsinto the analysis process. Therefore, and in order to minimize theseerrors, is necessary to standardize the input amount of RNA (orcDNA) and to simultaneously measure the levels of expression ofan endogenous RNA, which is expressed more or less in a constantmanner between the different samples or to employ an externalstandard [240,241].

The cellular genes used to normalize for the variability ofclinical samples or the integrity of the isolated nucleic acid in RNAprocedures, are genes involved in the regulation of basic andubiquitous cellular functions, and they are members of thehousekeeping gene family. They most widely used of them codefor components of the cytoskeleton (b-actin), major histocompat-ibility complex (b-2-microglobulin), glycolytic pathway (glycer-aldehyde-3-phosphate dehydrogenase, GAPDH andphosphoglycerokinase 1), metabolic salvage of nucleotides (hypo-xanthine ribosyltransferase, HPRT1), or synthesis of ribosomesubunits (rRNA) [240,241].

First of all, in the case of extracellular or circulating RNA, it iscrucial to evaluate the integrity of the isolated mRNA. This can besimply assessed by electrophoresis. In the cases, when a smallquantity of RNA is available this can be performed by PCRamplification using the selected control genes. We can postulatethat at least two or three of these genes can be selected in each casein order to obtain the best gene(s) standards. The choice can besimply done based on the biological sample used and the level ofexpression of the gene(s) of interest. In general, it was demon-strated that when only a small amount of RNA is available and thegene of interest exhibits a high level of expression, the high-expression GAPDH is the most suitable as an endogenous control

Table 1RNase inhibitors that are commonly used to stabilise RNA in the sample.

Agilent technologies Ambion Bioline Sigma-Aldrich Qiagen

RNase block RNAlater1 RiboSafe RNase inhibitor ProtectRNATM RNase inhibitor RNAlater

RNAlater1-ICE RNAprotect cell

SUPERaseIn RNAprotect saliva

Paxgene blood RNA

G. Tzimagiorgis et al. / Cancer Epidemiology 35 (2011) 580–589 583

gene with b-actin as a second choice [242]. Furthermore, de Koket al. argues that when the gene of interest has intermediate or lowexpression the best choice for the endogenous control gene is theHPRT1 gene, a low-copy housekeeping gene, which in combinationwith GAPDH, gives the best results in order to estimate RNAisolation efficiency, RNA quality, and RT-efficiency [243]. Otherstudies revealed that b-2 microglobulin and 18S rRNA are the mostsuitable internal control genes in quantitative serum-stimulationstudies, while b-actin and GAPDH are not so efficient [244]. On theother hand, some other studies proposed that the expression of theHPRT gene more accurately reflects the mean expression ofmultiple commonly used housekeeping genes [244]. Taking intoaccount all these considerations, we conclude that the internalcontrol gene(s) needs to be properly evaluated in order to selectthe most appropriate ones in a particular study.

4.5. RT-PCR or microarrays: advantages and disadvantages of

methods that can be used in extracellular RNA analysis

Analysis of gene expression, in general, can be done by severaldifferent methods including Northern blotting, RNase protectionassays, serial analysis of gene expression (SAGE), RT-PCR (andlately qRT-PCR), as well as with the microarray technology[245,246]. Among these techniques, although Northern blotanalysis and RNase protection assays are the ideal methods forthe measurement of gene expression/mRNA levels both require theuse of intact RNA, which rarely is the case in isolated extracellularRNA. On the other hand, the SAGE analysis is a highly complicatedand laborious technique, which also requires high quality RNA.Thus, the methods of choice to detect RNA species e.g. in plasma orserum is the techniques of RT-PCR or microarray analysis.

Microarray experiments allow for comparison of gene expres-sion profiles between two mRNA samples. There are twomicroarray platforms in common use cDNA microarrays, whichutilize cloned probe molecules corresponding to characterizedexpressed sequences, and oligonucleotide microarrays, made ofsynthetic probe sequences based on database information [247].Microarrays are quite commonly used and are usually consistentwith data obtained from northern blots. The major advantage thatmicroarrays have over all the other methods used in RNAexpression analysis is that thousands of genes can be visualizedat a time with a microarray, while all other techniques are usuallylooking at one or a small number of genes [247].

Real-time PCR is a type of quantitative PCR that can be used tocompare normal (control) samples to disease’s samples, providingclues as to the gene expression changes that occur in pathogenesis.The advent of real-time quantitative PCR (qRT-PCR) has eliminatedthe variability traditionally associated with quantitative PCR, thusallowing the routine and reliable quantitation of PCR products. Dueto its sensitivity, qRT-PCR, is the method of choice in order toquantify mRNA levels from even very small numbers of cells andlately even single cells or small amounts of biological fluids.Several advantages of this method over the microarrays are worthmentioning e.g. the low cost, the need of less expensive equipment,the relatively easy sample analysis. As with microarray analysis, inqRT-PCR, there is no need to work with intact RNA. Thus, althoughboth methods can be easily employed, the simplicity and thesensitivity of the qRT-PCR remain the major advantages.

5. Advantages and disadvantages of analyzing circulatingextracellular RNA instead of intracellular RNA

The analysis of extracellular circulating RNA is a powerful toolin order to evaluate a number of different pathological conditions,since it has proven to provide valuable information on theexpression pattern of several candidate genes [143,147]. Further-

more, the analysis is not limited to the RNA material released fromcirculating ‘blood’ cells but is also able to detect all circulating RNAoriginated from different ‘‘damaged’’ or ‘‘lysed’’ tissue cells, oftenexhibiting a malignant phenotype [5,19,20,42]. Moreover, theanalysis of cell free circulating RNA can provide information for thepresence of any abnormality without the need of material obtainedfrom biopsies, in the same way plasma/serum protein and enzymeanalysis is used in the diagnosis of several diseases andpathophysiological conditions. Furthermore, the analysis ofcirculating extracellular RNA can be employed easily in massscreening of high risk population [100,155,156] and in monitoringof metastases in cancer patients [51,69,89]. Additionally, theanalysis of extracellular RNA is the method of choice in order toavoid tedious and time consuming methods for the recruitment ofa number of circulating malignant cells in the blood in order toisolate and characterize the intracellular RNA content. Along thesame line, biopsies, the major source of intracellular RNA for geneexpression analysis studies, have as a prerequisite the presence of adisease diagnosis.

Despite the above-mentioned advantages, there are severaldisadvantages in working with extracellular RNA. The majority ofthese disadvantages arise from the low quality/quantity of theisolated RNA. Low RNA quality is a major obstacle in the analysis ofextracellular RNA. The isolated RNA if often degraded or fragmentedin small parts. As a consequence, different regions of the sametranscript might be represented differentially in the isolated RNA. Toovercome this problem there is a need to design carefully the PCRprimers in order to obtain the desired result. Often, there is a need todesign primer pairs detecting different regions of the transcriptunder investigation. In addition, and in order to have a roughestimation of the RNA quantity, it is necessary to use simultaneouslyas internal control, several genes (b-actin, b2m, GAPDH, 18S rRNA,etc.) [240,241]. Usually, the presence of amplification of at least twoof them in an RT-PCR reaction is required in order to select thesamples to be included in the study. Low RNA quantity, althoughmight be a potential problem, can be easily solved using real time RT-PCR protocols which are providing increased sensitivity. On thecontrary, the analysis of intracellular RNA, which is the alternativemethod, although it is free from all the above mentioneddisadvantages, request in all cases an invasive procedure (biopsy,fine needle aspiration, etc.) and in several circumstances to have adisease diagnosis.

6. Conclusions

Circulating extracellular or cell-free RNA obtained from avariety of bodily fluids can became a valuable tool in earlydiagnosis of several diseases including cancer. The major obstaclein order to be applicable for routine molecular diagnosticmethodology is to ensure the high quality and the integrity ofthe isolated RNA. In order to develop a reproducible methodologyof diagnostic value care should be taken in the use of the properRNA stabilizer in the clinical sample, in choosing of the appropriateRNA isolation procedure, ensuring the absence of any DNAcontamination, and finally, in the use of the most suitableendogenous housekeeping control genes, factors which arediscussed in detail in this article.

Conflict of interest statement

There is no conflict of interest.

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