in the last few years , a new class of highly conserved ~21-mer non - coding rnas , micrornas ( mirnas ) , has emerged as an important player in post - transcriptional gene expression control in different physiological and pathological conditions : metabolism , proliferation , cell death , differentiation and development , viral infection , and cancer [ 13 ] .
they specifically bind the 3-untranslated regions ( 3-utr ) of target mrnas , promoting either mrna degradation or translation arrest [ 46 ] .
each mirna may control the activity of many genes , and almost 30% of the genome could be regulated in such a way , which renders these small molecules as important as the transcription factors [ 7 , 8 ] . owing to their extreme importance as regulators , the isolation and the precise quantification of these tiny molecules are therefore fundamental . as purity and integrity are essential requirements not only for total rna but also for these small species , rna extraction protocols must also account for the maximizing of their recovery .
, it is well established that the traditional glass - fiber total rna extraction protocol may be inefficient at recovering small rnas .
in addition , the common spectrophotometric determination of total rna is not adequate to quantify low molecular weight ( lmw ) species selectively .
we and other authors found previously that the recovery of lmw rna species is significantly affected by the specific purification process [ 3 , 9 ] .
our results showed that different extraction strategies lead to significantly different recovery of lmw species including mirnas .
we also demonstrated that using the same amount of total rna ( from different tissues ) , different amounts of mirnas may be obtained .
moreover , the amount of lmw rna species does not perfectly parallel that of mirnas : even with the same extraction protocol , the concentration of mirnas may differ significantly among various tissues .
nevertheless , this may also be because of a different global expression of mirna by different tissues .
all of these considerations are extremely important when one compares the relative expression of many mirnas in different tissues .
commonly , some small non - coding rnas ( i.e. , u6 , snoz30 , and others ) are used as normalization controls to compare mirnas expression in different tissues .
the choice of a proper control should be conditioned to its real  endogenous behavior  , that is , it should have a constant and equal expression in all tissues .
if the amount of that control small non - coding rna is different , a relative quantification is not feasible . in this latter case ,
only the comparison between various mirnas and the control gene in the same specimen ( tissue ) is methodologically acceptable .
therefore , a method allowing a reliable estimation of the goodness of an endogenous control is highly desirable . in order to address all of these issues we used a lab - on - a - chip technology to characterize the lmw rna fractions obtained with different rna extraction protocols and evaluated mirnas recovery with specific quantitative real - time pcr ( qrt - pcr ) . in this paper
we report and discuss the methods that allowed us to analyze the efficiency of extraction protocols , to study the small ( but significant ) differences between various preparations and to obtain a correct comparison of some mirna expression profiles in various tissues .
total rna was extracted using three different methods : an acid phenol / guanidine isothiocyanate solution ( trizol reagent , invitrogen ) , a glass - fiber filtration protocol ( mirvana mirna isolation kit , ambion ) that provides also a procedure to isolate and enrich low molecular weight ( lmw ) rnas from higher molecular weight ( hmw ) rnas , and another common glass - fiber purification protocol ( rneasy mini kit , qiagen ) .
three different cell lines ( hela , cos-1 and a lymphoblastoid cell line ( lcl ) obtained following an already reported method ) were cultured using standard procedures , trypsinized ( if necessary ) , and pelleted by centrifugation .
approximately 10 cells for each extraction were resuspended in the appropriate lysis solution contained in the rna extraction kit and treated according to manufacturer 's instructions .
the integrity of rna samples was checked by gel electrophoresis ( agarose 1% ) stained with ethidium bromide .
gel images were acquired and analyzed with the quantity one ( software ver.2.0 , biorad ) .
two micrograms of each rna samples were resolved into a 15% polyacrylamide tbe - urea gel and the image acquired .
a second polyacrylamide gel , performed with the same experimental conditions , was used for rna bands extraction .
the visible bands were separately excised using a modified  crush - and - soak  method .
briefly , each band was gently disrupted with a pin and incubated in a soaking solution ( 0.5 m ammonium acetate , 2 mm edta , 0.1% sds ) for 3 hours at 37c .
the mixture was centrifuged at 14000 g for 5 minutes , and the supernatant was collected in a new tube .
the solution was centrifuged at 12000 g for 10 minutes through a ym-50 spin column , washed with water ( 3  300 l ) , and centrifuged at 12000 g for 5 minutes .
purified fractions were collected in new tubes by inverting the columns and spinning at 1000 g for 3 minutes .
the instrument uses fluorescence detection , monitoring the emission between 670 nm and 700 nm .
electropherograms were analyzed using the agilent 2100 expert b.02.06 software that includes data collection , presentation , and interpretation functions .
for the small rnas recovery efficiency evaluation of different extraction procedures , hsa - mir-21 and small nucleolar z30 ( snoz30 ) were assayed by real - time pcr ( taqman , applied biosystems ) , according to manufacturer 's instructions . for the analysis of mirna expression in different tissues ,
three mirnas ( hsa - mir-26a , hsa - mir-26b , and hsa - mir-134 ) and two controls ( u6 and snoz30 ) were arbitrarily chosen as model mirnas .
a panel of five tissues ( brain , skeletal muscle , heart , liver , and uterus ) was chosen for evaluation , and the corresponding total rna ( clontech , bd biosciences ) was analyzed with agilent 2100 bioanalyzer . briefly , from five to ten nanograms of each rna samples were retrotranscribed to cdna using the high - capacity cdna archive kit ( applied biosystems ) with specific primers .
the principle of taqman microrna assays is a   specific stem - loop reverse transcription ( rt ) primer .
the short length of mature mirnas ( ~22 nt ) prohibits conventional design of a random - primed rt step followed by a specific real - time assay . in the former case ,
the resulting rt amplicon is a suitable template for standard real - time pcr with taqman assays .
reactions were performed incubating samples for 30 minutes at 16c , 30 minutes at 42c , 5 minutes at 85c , and finally cooled on ice .
pcr products were assayed with specific probes using the taqman universal pcr master mix , no amperase ung ( applied biosystems ) following the manufacturer 's protocol .
pcr reactions were performed incubating samples for 10 minutes at 95c , then for 15 seconds at 95c and 60 seconds at 60c for 45 cycles by means of abi prism 7900ht sequence detection system .
. the early electropherogram region ( from 23 to 29 seconds ) , obtained from lcl rna sample extracted with trizol reagent , was exported in ascii format with the agilent 2100 expert b.02.06 and imported in peakfit v.4.12 .
the fitting procedure was iterated until a constant r value ( r = 0.998693 ) was obtained .
rna samples extracted from hela , cos-1 , and lcl were run on agarose gel to visualize the differences between various extraction methods .
cos-1 rna samples , extracted with trizol reagent and mirvana kit , clearly showed the high molecular weight ( hmw ) 28s and 18s rrna bands , while lmw rnas are visualized as faint , smeary bands ( figure 1 , lanes 1 and 2 , resp . ) .
cos-1 extracted with rneasy kit displayed only the hmw rna bands ( 28s and 18s ) ( figure 1 , lane 3 ) , while the enriched lmw fraction obtained with mirvana kit is clearly displayed in the lowest part of the gel ( figure 1 , lane 4 ) .
the same samples were also checked with agilent 2100 bioanalyzer which is one of the most versatile microfluidics - based platforms for the analysis of dna , rna , proteins and cells . in all electropherograms the 28s and 18s rnas are represented on the right side ( figure 2 ) , and the smaller species ( lmw rnas ) are present at a very low concentration and distinguishable on the left side of the profile ( see the magnification of the lmw rnas region in figure 2 ) . in the electropherogram ,
all samples present a similar hmw profile region , but the major differences are recognizable in the lmw region . while total rna recovery is quite good , and similar for the three protocols ( > 1.2 g/l ) , the lmw fractions are substantially different ( table 1 ) . in particular , trizol reagent allowed the highest lmw rna recovery ( 22%34% of total rna ) , while rneasy mini kit the lowest ( 2.5%3% ) .
mirvana mirna isolation kit gives good yields for lmw rna species ( 16%19% ) even before the enrichment step . while lmw rna species extracted with trizol and mirvana have comparable profiles ( figure 2 ) ; rneasy kit retains only one rna peak in comparable concentrations to the others ( peak a in the magnification of figure 2 ) .
therefore , the lab - on - a - chip analysis is a useful tool to quantify precisely the amount of lmw rnas of samples extracted with different protocols .
total rnas from lcl extracted with trizol reagent , and rneasy kit and enriched with mirvana kit were loaded on a polyacrylamide gel ( figure 3(a ) ) to analyze the differences between various extraction protocols .
all the rna bands were cut out from the gel , and the rna was extracted as described .
each species coming from excised bands was run on bioanalyzer , and the corresponding electropherograms ( figure 3(b ) ) were superimposed on the large and unresolved lmw rna profile ( black line ) .
this allowed us to localize unambiguously and identify precisely each rna species previously identified on polyacrylamide gel .
the electropherogram profile in the lmw rnas region is made of several peaks ( figure 3(b ) ) , the greatest part belonging to the 5.8s rrna ( red line ) , the 5s rrna ( green line ) , and the trnas fractions ( blue line ) . in order to obtain the principal number of peaks under the curve , peakfit software was used for the deconvolution of the lmw rna region .
seven peaks underlying the lmw rna profile were obtained with a good fit   ( r = 0.998693 ) .
the upper part of figure 3(c ) reports raw data ( ) together with the fitted curve ( solid line ) and the calculated baseline , while the lower part of figure 3(c ) shows the component peak functions .
calculated peak - peaking ( expressed in bp ) is also reported ( figure 3(c ) ) .
three of the calculated peaks centered at 145 bp ( 26.89 seconds ) , 131 bp ( 26.33 seconds ) , and 104 bp ( 25.24 seconds ) are located in the correspondence of 5.8s , 5s , and trna peaks , respectively .
another less intense peak is located at 114 bp ( 25.83 seconds ) and may be described as a further trna peak .
other peaks found at 171 bp ( 27.78 seconds ) , 89 bp ( 24.84 seconds ) and 65 bp ( 24.31 seconds ) might belong to other lmw rna species , such as smaller trnas , pre - mirnas , and small nuclear or nucleolar rnas .
these small peaks are difficult to extract from polyacrylamide gel owing to their low abundance , and therefore it was not possible to run them on the chip .
therefore , determination of small rna species by bioanalyzer ( table 1 ) and by polyacrylamide gel electrophoresis ( figure 3(a ) ) showed that the lmw rna samples significantly differ from each other . this could reasonably pertain also to the mirnas fraction . in order to quantify mirnas extracted with different protocols and to study the correlation between lmw rnas and mirnas , we carried out a taqman quantitative assay for two representative small rnas : a control non - coding rna ( snoz30 ) and a mirna ( hsa - mir-21 ) .
a target - specific stem - loop adapter technology was employed to obtain the corresponding cdna .
we started the rt reaction with 10 ng of total rna from each sample and quantified the absolute expression level of each mirnas through the analysis of cycle threshold   ( ct ) values .
ct is the pcr cycle at which the sample reaches the level of detection above the background .
lcl rna samples extracted with different protocols showed different ct values for snoz30 ( figure 4(a ) ) and for mir-21 .
a similar behavior was also obtained by using hela and cos-1 cell lines with both probes ( data not shown ) .
then , we repeated the rt reaction using 10 ng of lmw rna calculated from the bioanalyzer electropherogram .
as expected , using the same lmw rna amount similar ct values both for mir-21 and snoz30 between the various samples were obtained ( figure 4(b ) ) .
this demonstrates that the amount of mir-21 and snoz30 correlates only to the lmw rna fraction and not to total rna amount .
in fact , figure 4(a ) clearly shows that using the same amount of total rna different ct values may be obtained .
ct differences ( ct ) between total rna and lmw rna ct values for each extraction protocol and for each cell line are very similar both for hsa - mir-21 and snoz30 ( table 2 ) .
this demonstrates that ct differences parallel the amount of input rna that is ultimately linked to the amount extracted by various protocols .
table 2 reports also the calculated lmw rna concentration ( expressed in % ) calculated from ct values applying the formula 2 .
mirna concentrations calculated from real - time assays are , as expected , in perfect agreement with lmw rnas concentrations evaluated with the lab - on - a - chip technology . here
then , we asked if different tissues might also express different amounts of lmw and mirnas and if it might be possible to quantify them individually with lab - on - a - chip technology .
for this reason we selected five commercial rna samples from different tissues extracted with the same protocol ( according to manufacturer information ) .
total rnas from brain , skeletal muscle , heart , liver , and uterus were run on rna 6000 nano kit to quantify the total and the lmw rna fractions .
total rna concentrations were quite homogeneous and resulted : brain ( 1159 ng/l )   >   heart ( 1106 ng/l )   >   uterus ( 1080 ng/l )   >   liver ( 963 ng/l )   >   skeletal muscle ( 890 ng/l ) .
these values are in good agreement with the nominal manufacturer 's concentration of 1000 ng/l .
for all samples the concentration of lmw rna fraction was quite homogeneous and varied in the order : liver ( 43 ng/l )   >   brain ( 29 ng/l )   >   heart ( 26 ng/l )   >   uterus ( 25 ng/l )   >   skeletal muscle ( 23 ng/l ) .
electropherograms of lmw rnas of different tissues are displayed in figure 5(a ) . calculating the percentage of lmw rnas with respect to the whole total rna amount we found the following : liver ( 4.4% )   >   skeletal muscle ( 2.6% )   >   brain ( 2.5% )   >   heart ( 2.3% )   >   uterus ( 2.3% ) .
given that the extraction protocol employed by the manufacturer was the same for all samples , we could hypothesize that the only observed difference for liver might depend on the different global expression of small rnas in this   tissue .
to evaluate if the difference in lmw rna amount may also pertain to the mirna fraction , we ran the total rna samples on agilent small rna kit , specifically designed for the evaluation of mirnas . figure 5(b ) shows a magnification of the electropherogram profile obtained by running total rna samples from different tissues .
the displayed region ( from 35 to 45 seconds ) is specific to the mirna region ( as indicated by the manufacturer ) .
mirna concentrations were liver ( 700 pg/l )   >   brain ( 510 pg/l )   >   uterus ( 404 pg/l )   >   heart ( 226 pg/l )   >   skeletal muscle ( 71 pg/l ) . the mirna concentration , expressed in percentage , respect to lmw rnas resulted brain ( 1.7% )   >   liver ( 1.6% )   =   uterus ( 1.6% )   >   heart ( 0.9% )   >   skeletal muscle ( 0.3% ) . from this lab - on - a - chip quantification it was possible to conclude that the mirnas amount does not parallel that of lmw rnas in the same tissue . moreover , the variation is greater than that displayed by lmw rnas among different tissues . again , assuming that the extraction efficiency is the same for all samples , the observed differences are only due to the different global expression of mirnas in these tissues .
the small rna kit , specifically designed for the identification and quantification of mirna species , allowed us to know the exact amount of mirnas present in the samples used for retrotranscription ( rt ) reactions .
this information is useful , since the ct values obtained from real - time assays are directly proportional to the amount of mirnas .
in fact , the ct differences observed for the same mirna in different tissues may be due essentially ( 1 ) to a real difference between different samples and/or ( 2 ) to a different sample dilution that generates a shift in ct absolute values . to eliminate the dilution problem , that occurs when absolute quantification is performed , a relative quantification with respect to an endogenous control
in fact , the endogenous control must have a constant expression in all samples , and hence it may be used to normalize the expression of the other mirnas . for a methodologically correct comparison , however , one must be confident that the control ( i.e. , nuclear and/or nucleolar small rnas ) really does have a constant expression in considered samples .
the lab - on - a - chip technology that we used is able to give an estimate of this variation and let the researcher choose the right endogenous control ( the one that does not significantly vary ) from an adequate selection .
therefore , this validation ensures that the mirna expression comparison among considered samples is methodologically correct .
the expression of three mirnas ( hsa - mir-26a , hsa - mir-26b , and hsa - mir-134 ) and two small rnas ( u6 and snoz30 ) from the same five tissues analyzed before , was assayed with real - time pcr . according to the manufacturer 's suggestion , we started the rt reactions with 5 ng of total rna .
these values reflect the absolute concentrations of these mirnas in various samples . since most studies aim to discover differences in expression levels of mirnas and not absolute levels of expression , the use of an endogenous control is needed . in order to assess
if the differences in ct values of the controls we used ( u6 and snoz30 ) are because of a different starting concentration or a real differential expression , we corrected the obtained values by taking into account the concentration of mirnas previously obtained with the lab - on - a - chip technology .
hence , we considered that for double the concentration a correction of one ct value should be applied .
this preliminary correction eliminated the intrinsic variability owing to different sample concentrations and allowed us to estimate the reliability of the selected endogenous controls .
we observed slight variation for ct values of u6 ( average ct = 33.5  0.8 ) while snoz30 displayed a higher variability ( average ct = 34.1  1.1 ) even after the applied correction .
certainly , the lower the ct difference is , the more reliable are the results . to facilitate this ,
more lab - on - a - chip quantifications and real time assays are needed .
therefore , we concluded that u6 is a more reliable endogenous control than snoz30 for mirna expression profile comparison in the analyzed tissues .
figure 7 shows the expression values of hsa - mir-26a , hsa - mir-26b and hsa - mir-134 compared with the expression values of some tissue - specific mirnas ( mir-1 for heart and muscle , mir-122a for liver , mir-124a for brain ) .
one of the most difficult problems with mirnas experimental studies concerns the efficiency of their quantitative and qualitative recovery after total rna extraction from cells or tissues . in some cases , traditional total rna extraction protocols
are not efficient methods for extracting both high molecular weight ( hmw ) and low molecular weight ( lmw ) rnas .
moreover , conventional quantification methods ( i.e. , spectrophotometric measurements ) giving an overall quantification of total rna concentration are therefore inadequate for these small rna species .
northern blotting is the only current viable technique to visualize and evaluate small rna molecules , particularly mirnas .
however , their low abundance sometimes prevents the use of such techniques . on the other hand ,
the lab - on - a - chip technology we employed is an alternative and valuable tool for the precise quantification of small rnas and mirnas present in total rna samples . in this work , to characterize in detail all the lmw rna species visible in the bioanalyzer electropherogram ( 5.8s , 5s , trnas , etc . ) , we ran rna samples on polyacrylamide gels to clearly separate each of the predominant species present in the small rna region ( figure 3(a ) ) .
then , we monitored the eluted fractions by means of a lab - on - a - chip technology that is , at present , the only technique that allows for the comparison of small rna profiles and the exact quantification of these species ( figure 3(b ) ) .
several other species were present under the peak of lmw rnas , even if it was not possible to distinguish each of them . for these reasons
, we applied a mathematical algorithm ( the fourier transform ) for the deconvolution of the profile coming from the bioanalyzer into different gaussian peaks .
after the fitting procedure , we obtained a mathematical model comprising seven peaks that define the overall profile ( figure 3(c ) ) .
these peaks range from a minimum of 65 bp to a maximum of 171 bp , which is well in agreement with the results obtained with agilent automatic peak - peaking that relies on the comparison of rna species with an rna ladder included in the running buffer .
interestingly , after this procedure was performed some other non - assigned bands remained undetermined .
the small rna kit , specifically designed for the analysis of mirna species , was used to study these unidentified species in more detail .
we initially compared the three commonest rna extraction protocols , the classic acid phenol / guanidine isothiocyanate solution , and two glass fiber filtration protocols to examine and quantify the recovery of hmw and lmw rna species .
the acid phenol / guanidine isothiocyanate solution ( table 1 ) maximized not only the recovery of hmw rna fractions but also the lmw rnas ( from 22% to 36% ) , as previously observed .
then , we assessed by real - time pcr how significantly affected the recovery of mirna species was as a function of the total rna extraction protocol used . the expression levels of a mirna ( hsa - mir-21 ) and a small nucleolar rna ( snoz30 ) were evaluated by real - time pcr specific assays .
real - time quantification results ( table 2 ) demonstrated that the amount of mirnas correlates better with the amount of lmw rnas than with total rna .
another aspect that we critically analyzed concerned the correlation between lmw rna species and mirnas .
in fact , we proved that even with the same extraction protocol the concentration of mirnas may differ considerably between various preparations ( i.e. , extraction from different tissues ) .  
as a potential application of our results , we applied our findings to the comparison of some mirna expression in different tissues .
we assessed the expression of hsa - mir-26a , hsa - mir-26b , and has - mir-134 as model mirnas and two small rnas ( u6 and snoz30 ) as controls .
the expression profile comparison is correct only if we assume that the endogenous control ( u6 or others ) has a constant and equal expression in all the considered tissues .
the lab - on - a - chip technology we used allowed precise quantification of input mirnas , ultimately leading to a fine correction of real - time pcr ct data for eventual variations .
therefore , we obtained a reliable and correct estimate of the relative quantities of mirnas present in various tissues . surprisingly , the expression of mir-26a was the highest of all the mirnas in all the tissues considered ( fold change   >   10 with respect to u6 ) .
although our data are in good agreement with those reported in literature   ( figure 7 ) , the high expression values found for mir-26a will deserve future investigations .
however , the expression values of mir-26a reported in literature   are also very high ( and higher than those of mir-26b ) and above the values of those tissue - specific mirnas considered as highly expressed ( i.e. , mir-1 for muscle , mir-124a for brain , and mir-122a for liver ) ( see figure 7 ) . in conclusion
, all the methods we used in this paper allowed us to study accurately the efficiency of extraction protocols , analyze the small ( but significant ) differences between various preparations , and suggest a methodologically correct method for the comparison of mirna expression profiles in various tissues .
 micrornas ( mirnas ) are highly conserved 22-mer rna molecules , encoded by plants and animals that regulate the expression of genes binding to the 3-utr of specific target mrnas . 
 the amount of mirnas in a total rna sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed . 
 traditional approaches may be inefficient at recovering small rnas , and common spectrophotometric determination is not adequate to quantify selectively these low molecular weight ( lmw ) species from total rna samples . here 
 , we describe the use of qualitative and quantitative lab - on - a - chip tools for the analysis of these lmw rna species . 
 our data emphasize the close correlation of lmw rnas with the expression levels of some mirnas . 
 we therefore applied our result to the comparison of some mirna expression profiles in different tissues . 
 finally , the methods we used in this paper allowed us to analyze the efficiency of extraction protocols , to study the small ( but significant ) differences among various preparations and to allow a proper comparison of some mirna expression profiles in various tissues . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion