miRNA in the serum. Then a commercial kit was used to extract and isolate miRNA from the
exosomes. These steps ensure the yield and stability of miRNA and firmly underline the subsequent
experiments. Then we further validated through mass spectromery and proved that the RNAs in the
exosomes are mainly miRNAs which is consistent with the study of Valadi [24] . The miRNAs in the
exosomes are significantly richer than fresh serum. The serum without the exosomes still contain a
certain amount of miRNA, but the content is significantly lower than that in the exosomes. The
serum after repeated freeze-thaw was not found with any miRNA, but the exosomes thereby
isolated were still identified with a certain level of miRNAs. Afterwards, we used transcriptase-
polymerase chain reaction (RT-PCR) to detect three types of miRNAs in the exosomes. At present,
the methods for miRNA detection include Northern Blot, microarray chip, in-situ hybridization,
RTPCR, rolling circle amplification, and the use of conjugated polymers. Though microarray analysis
is able to analyze the miRNA expression profile, there are many novel chips and improved
techniques, but the sensitivity and specificity of microarray analysis should be further improved.
Various probe making techniques, amplification methods and detection methods should be further
integrated and improved. Analysis of in-situ hybridization shows that though the use of LNA probe
improves the analytical sensitivity and reliability, the effort and sensitivity of hybridization remain to
be improved. The sensitivity of new methods such as ISH should be further enhanced together with
in-situ amplification techniques. Though the existing miRNA amplification methods have pros and
cons, RT-PCR is the most widely-used amplification method, and there are many types of commercial
kits. Here we used RT-PCR into miRNAs detection. Compared with other methods, RT-PCR is superior
in terms of no-marking, simple operations, low cost, and high sensitivity, RT-PCR is able to get results
within short time, detect the expressions of multiple miRNAs, and even require less RNAs. Here RT-
PCR was successfully used to identify the expressions of mir-21, -133a and -181b in the serum
exosomes of colon cancer patients. We also prove that mir-21 is significantly higher expressed in
exosomes derived from colon cancer patients, especially in patients at the first diagnosis or before
any treatment. The mir-21 expression is about 5 folds than normal people. The difference is
significantly larger than the direct mir-21 extraction from the serum and facilitates early diagnosis.
Our study is almost limited by the relatively small number of cases of colon cancer patients;
however, our study showed that Exosomes can enhance the stability and integrity of miRNAs and
prevent miRNAs from RNA enzyme digestion and other environmental damage which maybe applied to clinical drug department.