Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration
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Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration
Approximately 10-15% of couples who want to conceive suffer from subfertility, while in 30% of these cases, male factors play a role. The level of specific microRNAs in seminal plasma, including those involved in spermatogenesis, can serve as a parameter indicative of subfertility. We first optimized protocols to obtain microRNAs from seminal plasma.
Next, using a test strategy is validated in a group of men, we aim to identify microRNAs whose levels associated with semen motility and concentration. By qPCR, 742 microRNAs are profiled in normozoospermic three samples, three samples of sperm and low sperm motility (asthenozoospermia), and three with semen low concentration (oligozoospermia). MicroRNAs show a significant difference between the groups further validated in a second group consisting of 40 samples with normozoospermia (control group), 47 samples with asthenozoospermia, and 19 samples with oligozoospermia (which is 74% too low motility).
MicroRNA highest results obtained with biofluids RNA extraction kits, with the inclusion of MS2 RNA and proteinase K treatment operator for the protocol, and when 50 uL seminal plasma is used as input. Exosome RNA isolation before extraction does not lead to improved results. In the test cohort, 236 microRNAs can be detected, of which 54 microRNAs showed differences between groups. Five microRNAs analyzed in the validation group. the level of MiR-34b-5p in the control group was significantly higher compared with asthenozoospermia group (p <0.05) and compared with oligozoospermia group (p <0.001).
We optimized the acquisition of seminal plasma microRNA and microRNA levels were identified in relation to sperm concentration and motility. As the latest human and rat studies showed that miR-34 family is a marker of low concentration of semen and is very important in spermatogenesis, seminal plasma miR-34b-5p may be a suitable candidate for further study as a marker of male subfertility.
Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration
When Less Is More: Special Capture and Analysis in the Plasma Tumor Exosom Liquid Biopsy Improves Sensitivity for Detection of Multiple androgen receptor Comprehensive Phenotypes in Advanced Prostate Cancer Patients
We evaluate the advantages and reliability of new protocols for enriching tumor extracellular vesicles (EV), allowing blood-based test for noninvasive parallel profiles of some of the androgen receptor (AR) gene changes. Three AR clinically relevant variants associated with response / resistance to standard-of-care treatment (AR-V7 transcripts, a point mutation AR T878A and AR gene amplification) were evaluated by digital PCR in 15 samples from patients affected by Castration-Resistant Prostate Cancer (CRPC).
Plasma is processed to obtain circulating RNA and DNA using protocols based enrichment EVS tumor through immuno-affinity and peptide-affinity compared with the generic extraction kit. Our results indicate that the immuno-affinity enrichment before RNA extraction clearly exceed the generic isolation method in the detection of the AR-V7, also allow for the differences between the respondents (R) and non-responders (NR) patients.The T878A mutation is detected, as a whole, in nine out of 15 samples, and no one approach alone is capable of expressing the mutation in all samples store, indicating that the method employed complementary.
Rat Complete SeraMir Exosome RNA Amplification and Profiling Kit for Media and Urine (contains cat# RA800TC-1 with 50ml ExoQuick-TC, RA805A-1 and RA812A-1 components)
AR amplification CRPC were detected in most samples were analyzed using either a cell-free DNA (cfDNA) or exosome isolation kit (80%). We show that the selective isolation of exosomes enriched subset for the origin of the tumor, rather than the total analysis of exosomes plasma or plasma total nucleic acids, increased sensitivity and specificity to detect specific changes in circulation.
