the interest in understanding the genetic basis of diseases and drug regimens has increased , requiring dna isolation . however the extraction of dna from buccal cells ( bc ) is bringing a new perspective to obtaining dna8 . compared to other methods , such as blood collection , it is a noninvasive collection method and is therefore better tolerated by adults , children and handicapped individuals . this procedure has low cost and does not require medical assistance ( e.g. : nurse ) , so a wide population can be genotyped1,3,6,9 . the wet method consist in swishing liquids in the mouth and spitting them into a collecting cup2 . this procedure yields a higher amount and longer fragments of dna2,4 , requires more steps , is more cumbersome and has a higher cost8 . they are simpler , more cost effective8 and are considered less sensitive to the effect of long time storage at room temperature when compared to mouthwash , which may be crucial for multicenter studies6 . according to a recent study , the use of cytobrushes appears to be the most appropriate manner to facilitate self - collection of human genomic dna with good quality and high security when compared to the mouthwash method6 . commercial kits are available to extract dna from bc and they can provide sufficient dna for genetic analysis . nevertheless , due to logistic , financial , practical and methodological reasons , large studies often require the storage of bc samples before dna extraction7 . there is a lack of information in the literature regarding the best method to store bc after collection . therefore , the purpose of this study was to evaluate quantitatively and qualitatively the effect of the storage time of samples before the application of the cell lysis solution ( cls ) for extracting dna from bc . the research protocol was reviewed and approved by the research ethics committee of the federal university of pelotas , brazil . five volunteers were selected to participate in the study and were submitted to three bc collections . in each collection , 2 bc samples were obtained , being one from the upper and one form the lower gutter region . samples were collected using disposable special cytological brushes and the volunteers were instructed to brush and twirl each cytobrush for 30 s over the gutter region , which is the space between the gums and the inner portion of the lips and cheeks along the front and sides of the mouth . for all volunteers , the collections were performed in different anatomical regions of the oral cavity at the same time , with 5 days of interval between each collection . the collected samples were treated in three different forms . in the control group ( n=10 ) , immediately after the collection , the cytological brush containing the material was introduced in a microfuge tube that contained cls . after 3 h in this solution , the samples were submitted to laboratory processing for dna extraction . in the second group ( n=10 ) , soon after collection , the cytobrush was placed in a microfuge tube , without cls , and left undisturbed for 72 h at room temperature . thereafter , cls was applied and the samples were processed in the same way as in the control group . in the third group ( n=10 ) , the collected samples remained in the microfuge tube for 72 h at 4 c. after this period , cls was applied and the samples were processed in the same way as in the control group . all samples were processed following the manufacturer 's instructions ( puregene dna buccal cell kit ; gentra systems , inc . , minneapolis , mn ) . according to the manufacturer , 0.2 to 2.0 g / brush of dna after processing of each sample , 20 l of solution was obtained . from this amount , 10 l were placed in a tube containing 90 l of milli - q water and the amount of dna was read in a spectrophotometer ( eppendorf biophotometer , hamburg , germany ) , in which dna concentration and purity was evaluated . the average from the two collections in each individual was obtained , with samples submitted to the same storage condition and the obtained data analyzed statistically by one - way anova . in order to ascertain the presence of high molecular weight dna in the samples , the remaining 10 l of each sample were used to run 0.8% agarose gel electrophoresis at 2v / cm and were stained with ethidium bromide . the dna degradation was observed by fragmentation of the samples compared against a known molecular weight marker . the visible bands were examined by two calibrated examiners that were not involved in the study . the means and standard deviations for dna extracted under immediate , room temperature and cooling temperature conditions were 3.5  0.7 , 3.0  0.6 and 4.1  1.8 g , respectively . the statistical analysis did not show statistically significant difference regarding dna concentration among the three storage conditions ( p=0.385 ) . regarding the qualitative evaluation , differences in band patterns processed immediately after bc collection , no degradation traces were observed ( figure 1 ) . however , in the others groups with samples processed later under room and cooling temperature conditions , dna degradation or absence of bands were observed ( figure 1 ) . this study is particularly relevant for large - scale epidemiological studies , where generally the laboratory is not available to process the genetic material immediately after collection in a field study . therefore , sample storage is frequently necessary to prevent the loss of quantity or quality of the genetic material . although bc samples give a smaller amount of dna then blood samples , recently developed methods of genotyping use very small amounts of dna what makes the collection of bc a viable source of genetic material with high quality . furthermore blood collection is more invasive and not well tolerated by some patients , making easier the compliance in larger epidemiologic studies4,9 . the amount of dna material obtained from bc is dependent of the location , the force applied during collection and the patients ' individual variations6 . another factor that is capable to influence the amount of material obtained is the time of collection . in the present study , the time of collection of 30 s this time was used by several studies3,9,11 since longer times are unviable , especially in studies involving children . in order to avoid loss of material in the present study , the collections were made before volunteers had eaten or brushed their teeth , since the attrition of foods and toothbrush could reduce significantly the amount of material on the mucous membranes . the selection for the gutter region of the mouth instead of the inner cheek was based in a recent study by saftlas , et al.9 ( 2004 ) , where collection with cytobrushes in adult women from the gutter provided significantly larger amounts of dna ( 7.5 g ) than the standard method of brushing the inner cheeks ( 3.8 g ) . however , in the present study , the mean amount of dna obtained from the gutter area was 3.5 g , which is similar to the quantity obtained from the inner cheeks in saftlas ' et al . study . in addition , the value obtained in the present study is similar to that reported by mulot , et al.6 ( 2005 ) using cytobrushes that were twirled in the inner cheek during 15 s. the same author observed that there was no significant decrease of the dna yield between 2 , 5 and 7 days , while all samples were maintained at room temperature6 . these results corroborate those of the present study , with no significant difference between the three methods of storage after material collection . in addition , for all methods , the amount of dna was greater than 1 to 2 g , which is considered adequate for epidemiological studies6,9 . the qualitative evaluation was based on the 0.8% agarose gel observation . a decrease in dna quality was observed when the material was not immediately placed in cls and processed , but was rather stored at room temperature or kept refrigerated until immersion in cls and processing . this could suggest that soon after collection of genetic material , the brushes should be placed immediately in the lysis solution to preserve the material 's quality . king , et al.5 ( 2002 ) verified the dna quality using pcr amplification and observed that cytobrushes collections , contains dna fragments for short and intermediate amplification primers , and poor results for large gene fragments . according to the authors , it might have occurred due to degradation of the dna from the cytobrushes , suggesting that the period of storage is an important issue in dna quality , which was reaffirmed by the findings of present study . however , we also observed that the degradation process could be minimized by immediately placing the samples in the cls . finally , it is important to highlight that genetic material collection with cytobrushes has proved to be an effective and low cost method for the collection of genetic material for epidemiologic studies . within the limitations of this study it may be concluded that the amount of dna obtained was not influenced by the method of storage prior to cls application . however , the quality of the genetic material was more preserved when cell lysis was performed immediately after sample collection .