Recovery Of Cell-free MRNA And MicroRNA From Human Semen Based On Their Existing Forms

Background and Objective:In our previous research, it has been found that human cell-free seminal mRNAs (cfs-mRNAs) are substantially present in the seminal microvesicles (SMVs), which protecting seminal plasma mRNA against seminal ribonuclease activity. Since the diameter of most SMVs are above0.10μm, in the present study a microfilter (0.10μm) was used to collect SMVs for cfs-mRNA recovery, aiming to develop a method for cfs-mRNA isolation based on its existing form.Methods:SMVs were collected from semen of normozoospermic individuals by the microfiltration (MF) of the0.10μm filter and subjected to the Trizol LS reagent (Invitrogen) procedure to extract cfs-mRNA. Cfs-mRNA quantity was assessed by real time RT-PCR targeting transcripts of a house keeping gene (β-actin, ACTB), testis specific genes (DEAD box polypeptide4, DDX4;Protamine2, PRM2), an epididymis specific gene (WAP-type four-disulfide core domain9, WFDC9), a prostate specific gene (serpin peptidase inhibitor, clade A, member5, SERPINA5), a seminal vesicle specific gene (transglutaminase4, TGM4). Quantities of cfs-mRNAs were compared with single Trizol LS reagent procedure without0.10μm filtration, to analyze the impact of0.10μm membrane to the yield of cfs-mRNA. Results:Comparing with the cfsRNA extracted from the unfiltered seminal plasma,93.9%15.7%(Mean±SE) ACTB,99.1%±12.5%DDX4,76.9%±18.5%PRM2,88.1%±29.6%WFDC9,150.2%±73.6%TGM4,98.6%±17.8%SERPINA5were recovered from the membrane of the0.10μm microfilter.The yield of cfs-mRNAs recovered from the membrane was comparable with total amounts in seminal plasma (P>0.05, n=6, Paird-Samples/-test).Conclusion:The extraction method based on the presence forms,utilizing0.10μm filter membrane to enrich the SMVs and then extracting mRNAs, does not result in a significantly reduce of mRNAs yield. Also, the experimental results are consistent with our previous study about the SMVs and cfs-mRNAs. Background and Objective:The above RNA extraction method based on the presence forms does not result in a significantly reduce of mRNAs yield and is easy to operate. We further presumed that it should improve the purity of the mRNA. The present experiment aimed to analyze the purity of cfs-mRNA, and DNA contamination.Methods:The purity of the extracted RNA was measured by a spectrophotometer (OD260/280). And the distribution of RNA fragments was analyzed by capillary electrophoresis. The residual amount of DNA in cfs-mRNAs was assessed by quantitative PCR (qPCR) targeting DDX4DNA. Also, we detected the amount of the filtrate and filter DNA to analyze the impact of the membranes for DNA.Results:Compared with cfsRNA extracted from the unfiltered seminal plasma, the OD260/280value of our new method (1.828±0.051, n=6) increased significantly (P<0.05, Paird-Samples t-test). Capillary electrophoresis showed that in microfiltration group the proportion cfsRNA above500nt increased than unfiltered group. Determined by qPCR, DNA residues in cfs-mRNA significantly reduced in the new method. The DNA concentration in RNA of microfiltration group was15.5±10.3ng/ml, whearas in the unfiltered group was216.4±95.2ng/ml. DNA substantially present in the filtrate after microfiltration through0.10μm membranes in the process of DNA extraction from the filtrate and filtration. Yield of DNA was set to1in the unfiltered seminal plasma. Relative to the unfiltered group, the proportion of DNA amount in the MF membrane was14.4%and162.6%in the filtrate.Conclusion:The0.10μm filter membrane can efficiently enrich the SMVs. DNA residues are reduced and purity is improved apparently. And large amounts of DNA can be filtered through0.10μm membrane. This is more conducive to the extraction of the cfs-mRNA on the filter. Background and Objective:In the previous study, the vast majority of human cell-free seminal microRNA (cfs-miRNA) was found to be existed in the form of protein complexes, which is less than0.10μm in diameter. Therefore, the present experiment aimed to extract cfs-miRNA from protein complexes which is enriched from the filtrate of the0.10μm microfiltration by ultrafiltration (UF).Methods:Seminal plasma was filtered through the0.10μm microfilter. And the filtrate was centrifuged by a30kDa protein ultrafiltration(UF) tube. Then cfs-miRNA was recovered from the protein concentrate. Quantity of cfs-miRNA was assessed by real time RT-PCR targeting miRNAs with high abundance in semen (miR-30d, miR-93), or with specific expression in the male reproductive organs (miR-202, miR-514, miR-891b, miR-892a, miR-141, miR-221), and compared with their amounts in total cfsRNA which was extracted from seminal plasma by Trizol LS Reagent.Results:Compared with miRNA levels in total cfsRNA extracted from seminal plasma, the relative yield of cfs-microRNAs recovered from the protein concentrate were119.1%±57.2%(miR-30d),165.1%±62.3%(miR-93),108.6%±23.3%(miR-202),126.7%±45.4%(miR-514),163.4%±88.0%(miR-891b),142.1%± 54.6%(miR-892a),129.7%±59.9%(miR-141),94.9%±13.2%(miR-221). No significant difference was observed in miRNA levels between the total cfsRNA and the cfs-miRNA recovered from the protein concentrate (P>0.05, Paired-Samples t-test).Conclusion:30kDa protein concentrate centrifugal can enrich the protein complex particles containing cfs-miRNAs. The method based on the cfs-miRNA existing form can effectively extract cell-free miRNA from human seminal plasma. Background and Objective:From the data of above experiment, we find that the extracted miRNAs in the protein concentrate have no significant decrease. They are enriched based on their existing forms with the use of ultrafiltration centrifuge tube for concentrating protein, indicating the feasibility of this extraction method. Whether this method, which is able to enrich and extract microRNA/protein complexes, can improve the quality of the miRNA? The present experiment determined the RNA quality of this protocol.Methods:The distribution of RNA fragments was determined by capillary electrophoresis. The residual amount of DNA in cfs-miRNA was assessed by qPCR targeting DDX4introns. Also, the DNA amount in protein concentrate and the filtrate was quantified by qPCR. Results:Similar with cfs-miRNA, most genomic DNA was present in protein concentrate, and little amount DNA passed the ultrafilter. Yield of DNA was set to1in the unfiltered seminal plasma. Relative to the unfiltered group, the proportion of DNA amount in the UF tube was237.0%and0.8%in the filtrate. However, DNA residues in cfs-miRNA extracted by our new method (176.5±110.4ng/ml) were significantly reduced compared with the DNA concentration (281.9±159.9ng/ml) in total cfsRNA isolated from the unfiltered seminal plasma. Capillary electrophoresis showed that in ultrafiltration group the proportion cfsRNA below200nt increased than unfiltered group.Conclusion:The30kDa ultrafiltration centrifuge tube can efficiently enrich the miRNA/protein complexes. DNA residues were reduced and purity is improved apparently.