Male-specific Markers Research Articles

Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix).

Sex-specific markers are powerful tools for identifying sex-determination system in various animals. Bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix) are two of the most important edible fish in Asia, which have a long juvenility period that can lasts for 4–5 years. In this study, we found one sex-specific marker by next-generation sequencing together with bioinformatics analysis in bighead carp. The male-specific markers were used to perform molecular sexing in the progenies of artificial gynogenetic diploids and found all progenies (n = 160) were females. Meanwhile, around 1 : 1 sex ratio was observed in a total of 579 juvenile offspring from three other families. To further extend the male-specific region, we performed genome walking and got a male-specific sequence of 8,661 bp. Five pairs of primers were designed and could be used to efficiently distinguish males from females in bighead carp and silver carp. The development of these male-specific markers and results of their molecular sexing in different populations provide strong evidence for a sex determination system of female homogametry or male heterogametry (XX/XY) in bighead carp and silver carp. To the best of our knowledge, this is the first report of effective sex-specific markers in these two large carp species.

Identification and characterization of sex-specific markers in the milky mangrove Excoecaria agallocha using double digest restriction site-associated DNA sequencing

Sex-specific markers can be used to infer sex determination systems of dioecious plants and to study the evolution of plant sex determination. Meanwhile, sex identification of plants with agricultural or economic value using sex-specific markers can be beneficial for rapid breeding practice and better commercial harvest. Although there are more than 15,000 dioecious flowering plants, the sex determination systems of less than 40 species have been identified. A major barrier to studying plant sex determination and evolution is the lack of information on the types of sex determination mechanisms. In this study, we used double digest restriction site-associated DNA sequencing (ddRAD-seq) to identify sex-specific markers in a dioecious mangrove tree, Excoecaria agallocha. We performed ddRAD-seq for multiple females and males of E. agallocha in a natural population and identified six candidate male-specific loci among 177,071 catalogue loci for these samples. In contrast, no female-specific loci were found among these catalogue loci. We further validated two of the six male-specific loci using PCR on the individuals used for ddRAD-seq and additional individuals from another geographically distant population. Our results indicated that E. agallocha has a genetic sex determination system. We found that sex ratios in the seeds and one-month-old seedlings of E. agallocha didn’t deviate from 1:1 by using PCR amplification of one male specific locus, suggesting post-young-seedling selection may cause skewed sex ratio towards adult males. Reliable sex identification of seedlings of E. agallocha with male-specific markers is valuable for rapid recovery of mangrove forests. The ddRAD method developed here can also be used to identify sex-specific markers in other dioecious plants, especially for trees in which experimental crosses are impractical.

A genome screen for the development of sex-specific DNA markers in Saccharina japonica

Saccharina japonica is an important cultured marine brown alga and it has been the subject of intensive genetic improvement during its domestication and farming. However, there are limited credible and effective sex markers to identify the sex of gametophytes and sporophytes derived from normal hybridization or monogenesis. In the present study, the transcriptomic and genomic sequences of S. japonica were screened by 15 sex-determining region (SDR) genes from a model brown alga (Ectocarpus), and six cDNA sequences and their corresponding genomic sequences were identified. Thirteen primer pairs were designed from the coding region of six genomic sequences and subsequently tested on gametophytes of S. japonica. From these regions, four novel male-specific markers were developed, which derived from S. japonica genomic sequences JXRI01001736 and JXRI01002827, corresponding to a male-specific SDR gene and a gametologue SDR gene in Ectocarpus, respectively. In addition, an existing female marker (M_68_58_2) for Macrocystis pyrifera and Undaria pinnatifida, derived from the Ectocarpus SDR gene, could also be used as a female-specific marker for S. japonica. The results of the present study not only demonstrate an important new tool to determine the sex of gametophytes and distinguish between diploid sporophytes and partheno-sporophytes in breeding programmes but also provide insight into the sex-determination system for S. japonica, including the identification of sex chromosomes, SDR, and SDR genes and evolution of sex chromosomes.

A method for sex identification in asparagus using DNA from seeds

Asparagus (Asparagus officinalis L.) is a dioecious species, with both male and female individuals. Male plants are more desirable to cultivate than female plants because they have higher yields, and, unlike female plants, they do not have a weed problem resulting from fallen seeds. A male-specific DNA marker is currently available to identify the sex of asparagus individuals using total DNA extracted from cladodes and roots. However, no published method is currently available for DNA extraction and PCR amplification from asparagus seeds. In this study, we tested several heat-resistant DNA polymerases for PCR and several methods for extracting DNA from asparagus seeds and successfully established a method for identifying the sex of asparagus seeds using this male-specific DNA marker. We found that PCR amplification of DNA extracted from asparagus seeds using simple methods such as single-step DNA extraction requires the use of high efficiency DNA polymerase. By contrast, many types of heat-resistant DNA polymerases can be used for PCR amplification of high-quality DNA extracted from asparagus seeds using a commercially available DNA extraction kit. Our method for sex identification of asparagus seeds could facilitate quality checking of all-male asparagus seeds and accelerate the screening of super-male asparagus.

Identification of a male-specific DNA marker in the large yellow croaker (Larimichthys crocea)

Understanding sex determination mechanisms is the first and most paramount step in the development of sex control breeding biotechnologies for fish. The large yellow croaker (Larimichthys crocea) is an economically vital fish in China and East Asia, which exhibits a sex-related growth dimorphism where females grow significantly faster than males. However, the lack of a rapid and accurate sex specific marker for the large yellow croaker has hampered the application of sex control in the breeding process, as well as sex-determination investigations of this species. In this study, a male-specific 15bp deletion in the third intron of the Dmrt1 gene was identified by comparative analysis of genomic re-sequencing data from male and female samples. Two pairs of primers (MS and MFS) were carefully designed; thus, the male-specific marker could be genotyped by PCR amplification and agarose gel electrophoresis. More importantly, this male-specific marker could also be applied to precisely identify the genetic sex of multiple fish. This male specific DNA marker will have potential applications in the elucidation of the sex determination mechanism and will also accelerate the development of sex control breeding in the large yellow croaker. Moreover, our current data further validated an significant genetic evidence that an XY-male determination mechanism is utilized in the large yellow croaker.

A study of the Bodrogköz population in north-eastern Hungary by Y chromosomal haplotypes and haplogroups.

We have determined the distribution of Y chromosomal haplotypes and haplogroups in population samples from one of the most important areas in north-eastern Hungary from many villages in the Bodrogköz. The Bodrogköz region was chosen due to its isolated nature, because this area was a moorland encircled by the Tisza, Bodrog, and Latorca Rivers and inhabitants of this part of Hungary escaped from both Tatar and Ottoman invasions, which decimated the post-Hungarian Conquest populations in many parts of the country. Furthermore, in the first half of the tenth century, this region served as the Palatial Centre and burial grounds of the Hungarian tribes. It has thus been assumed that the present population in this area is likely to be more similar to the population that lived in the Conquest period. We analysed male-specific markers, 23 Y-STRs and more than 30 Y-SNPs, that reflect the past and recent genetic history. We found that the general haplogroup distribution of the samples showed high genetic similarity to non-Bodrogköz Hungarians and neighbouring populations, despite its sheltered location and historical record. We were able to classify the Y-chromosomal haplogroups into four large groups based on STR mutation events: pre-Roman/Roman ancient lineage, Finno-Ugric speakers arriving into the Carpathian Basin, Migration period admixture, and post-Hungarian Conquest admixture. It is clear that a significantly larger database with deep haplogroup resolution, including ancient DNA data, is required to strengthen this research.

Male-specific molecular genetic markers in the Japanese subterranean termite Reticulitermes speratus

Sex-specific genetic markers are often required for studying sex-associated phenomena. Restriction site-associated DNA sequencing (RAD-seq) allows detection of a huge number of genetic polymorphisms and is particularly useful for identifying sex-specific DNA-based (or molecular) genetic markers. Although sex determination in the Japanese subterranean termite Reticulitermes speratus depends on an XX/XY chromosome system, in which male-specific molecular genetic markers can be developed, no such marker has been developed. In this study, we developed male-specific genetic markers for R. speratus using RAD-seq with two restriction enzymes (i.e. double digest [dd] RAD-seq). We subjected 58 field-collected individuals per sex to ddRAD-seq and obtained around 210 million paired-end reads. Our in silico analysis of ddRAD-seq data detected 25 male-specific loci but no female-specific loci, thus confirming an XX/XY system. To verify the male specificity of those loci, we conducted PCR with primers designed for male-specific loci. Accordingly, we obtained male-specific PCR amplifications in six of the 25 loci. Furthermore, we examined whether these six male-specific markers were also applicable to individuals derived from four Japanese populations of R. speratus, including from the northern and southern ends of the distribution range in Japan. Of the six markers, five exhibited male-specific PCR amplifications in all four populations; the sixth was applicable to three populations. PCR amplification tests for cross-species applicability in R. kanmonensis found that none of these markers were cross-species applicable. The six male-specific markers would be useful for sex and sex chromosome identification in R. speratus individuals at all life stages.

Development of a sequence-tagged site (STS) marker for sex identification in the dioecious rattan species Calamus guruba Buch.-Ham.

Calamus guruba Buch.-Ham., a perennial, dioecious rattan species of the family Arecaceae has recently emerged as an important source of tribal economy owing to its high-quality flexible canes for furniture manufacturing and cottage industries. The dioecious nature together with disproportionate distribution of male and female sex organs has been the major constraint in the development of an effective breeding programme for the plant. Early identification of the male and female genotypes at the seedling stage is a pre-requisite to ensure genetic improvement of C. guruba. In the present study, we used 30 inter simple sequence repeat (ISSR) markers to develop a sequence-tagged site (STS) linked to male sex in C. guruba. Molecular analysis of bulked DNA pooled from male and female genotypes resulted in the isolation of a putative male-specific marker CgMSM. Partial sequencing of CgMSM resulted in an STS marker CgMY1 which could successfully amplify a 597-bp fragment in male but not in the female plants. DNA gel blot analysis confirmed it as a single-copy locus in the male genome of C. guruba. Further validation of CgMY1 in the natural population of C. guruba resulted in precise detection of 9 males and 6 females from 15 individuals with unknown sex. Therefore, the STS marker CgMY1 could be used as a proficient tool for early sex differentiation and form the basis of a sustainable breeding programme for genetic improvement of C. guruba.

Sensitive DIP-STR markers for the analysis of unbalanced mixtures from “touch” DNA samples

Casework samples collected for forensic DNA analysis can produce genomic mixtures in which the DNA of the alleged offender is masked by high quantities of DNA coming from the victim. DIP-STRs are novel genetic markers specifically developed to enable the target analysis of a DNA of interest in the presence of exceeding quantities of a second DNA (up to 1000-fold). The genotyping system, which is based on allele-specific amplifications of haplotypes formed by a deletion/insertion polymorphism (DIP) and a short tandem repeat (STR), combines the capacity of targeting the DNA of an individual with a strong identification power. Finally, DIP-STRs are autosomal markers therefore they can be applied to any combination of major and minor DNA.In this study we aimed to assess the ability of DIP-STRs to detect the minor contributor on challenging “touch” DNA samples simulated with representative crime-associated substrates and to compare their performance to commonly used male-specific markers (Y-STRs). As part of a comprehensive study on the relative DNA contribution of two persons handling the same object, we selected 71 unbalanced contact traces of which 14 comprised a male minor DNA contributor mixed to a female major DNA contributor. Using a set of six DIP-STRs, one to four markers were found to be informative for the minor DNA detection across traces. When compared to Y-STRs (14 traces), the DIP-STRs showed similar sensitivity in detecting the minor DNA across substrate materials with a similar occurrence of allele drop-out. Conversely, because of the sex combination of the two users of the object, 57 remaining traces could only be investigated by DIP-STRs. Of these, 30 minor DNA contributors could be detected by all informative markers while 12 traces showed events of allele drop-out. Finally, 15 traces showed no amplification of the minor DNA. These last 15 samples were mostly characterized by a combination of short handling time of the object, low DNA recovery and/or one single informative DIP-STR.In conclusion, the DIP-STRs represent alternative markers to help solving unbalanced two-source DNA mixtures, and also those produced from contact stains. These markers, in addition to a novel set of 10 DIP-STRs specifically developed according to forensic technical standards, will offer a valuable tool complementary to Y-STR markers.

Development of male-specific markers and identification of sex reversal mutants in papaya

Papaya is a productive and nutritious fruit grown in tropical and sub-tropical regions worldwide. It is polygamous with three sex types: female, male and hermaphrodite. Sex determination in papaya is controlled by an XY sex chromosome system with two slightly different Y chromosomes, Y for males and Yh for hermaphrodites. Comparative analysis of the hermaphrodite-specific region of Yh chromosome (HSY) and male-specific region of Y chromosome (MSY) revealed 99.6% sequence identity, which explains why DNA markers that amplify for both males and hermaphrodites have easily been developed, but not for the male trait specifically. We examined the 0.4% sequence differences, and found 1887 indels and 21,088 SNPs between MSY and HSY. The vast majority of indels are single nucleotide or few base pairs. A large male-specific retrotransposon insertion of 8396 bp was used to develop two papaya male-specific markers, PMSM1 and PMSM2 that amplify 585 and 548 bp fragments, respectively. These two markers were tested in 11 gynodioecious and four dioecious varieties along with autosomal DNA marker 71E and male/hermaphrodite marker W11, and the results showed clear separation of male from hermaphrodite and female. PMSM1 and PMSM2 were also used to test the sex type of six sex male-to-hermaphrodite reversal mutants which are crucial materials for validating candidate genes for sex determination in papaya. Our result showed all six mutants were positive for the male-specific markers. These male-specific markers can be used to distinguish gynodioecious and dioecious cultivars in papaya seed market, and facilitate genetic and genomic research for papaya improvement.

Rapid sex identification method of spinach (Spinacia oleracea L.) in the vegetative stage using loop-mediated isothermal amplification.

A LAMP-mediated, simple and rapid method for sex identification in spinach was developed. Nutrient compositional analysis showed a higher iron content in male than female plants. Spinach (Spinacia oleracea L.) is a dioecious plant with its sex determined by the XY system. Male and female floral organs differ morphologically, but plants do not differ in the vegetative stage before flowering. PCR with Y chromosome markers has been used to determine the sex of dioecious plants before flowering. In this study, we developed a genotype-specific loop-mediated isothermal amplification (LAMP) for sex identification of individual vegetative-stage spinach plants, using primers designed for the genomic region flanked by male-specific markers. LAMP could specifically detect spinach males. The method was further modified to omit DNA purification and use just an aliquot of crude leaf extract homogenized in water. We compared the nutrient composition of males and females, finding higher amounts of iron in the males. Our method could therefore be used for rapidly discriminating male plants in the field, which is useful for efficient hybrid breeding.

Extra Microchromosomes Play Male Determination Role in Polyploid Gibel Carp.

Sex is generally determined by sex chromosomes in vertebrates, and sex chromosomes exhibit the most rapidly-evolving traits. Sex chromosome evolution has been revealed previously in numerous cases, but the association between sex chromosome origin and the reproduction mode transition from unisexual to sexual reproduction remains unclear. Here, we have isolated a male-specific sequence via analysis of amplified fragment length polymorphism from polyploid gibel carp (Carassius gibelio), a species that not only has the ability to reproduce unisexually but also contains males in wild populations. Subsequently, we have found through FISH analysis that males have several extra microchromosomes with repetitive sequences and transposable elements when compared to females. Moreover, we produced sex-reversed physiological females with a male-specific marker by using estradiol hormone treatment, and two gynogenetic families were established from them. In addition, the male incidence rates of two gynogenetic families were revealed to be closely associated with the extra microchromosome number of the sex-reversed physiological females. These results suggest that the extra microchromosomes in males might resemble a common feature of sex chromosomes and might play a significant role in male determination during the evolutionary trajectory of the reproduction mode transition from unisexual to sexual reproduction in the polyploid fish.

A male-specific SCAR DNA marker and sex ratio of seedlings in salak (Salacca zalacca var. zalacca)

A male-specific SCAR DNA marker was developed using a RAPD DNA marker specific for male plants of Salacca zalacca var. zalacca (salak palm). The marker is 1579 bp long and has a GC content of 38.5 %. Its sequence contains 1 or 2 open reading frames, indicating the marker is probably a coding region. No highly similar sequences were found in a search of the GenBank database. Sexes were identified using the SCAR DNA marker for three kinds of seedlings grouped by the number of seeds per fruit (1, 2 or 3). The sex ratio of female to male did not differ significantly from 1:1 for the three kinds of seedlings, implying that the number of seeds per fruit is not a reliable index to identify the sex of a seedling.

ISSR and SSR markers reveal sex-specific DNA sequences in three Calamus species from India

Calamus species yields the raw materials for the cane industry. However, the extraction of the cane from the forests is being carried out indiscriminately without considering the sustenance of the species. Plants of both sexes should co-exist for reproductive success. The sex of Calamus plants can be identified only after flowering and hence proper planning of managed forestry practices cannot be accomplished. A study was carried out in this background and male specific ISSR markers for C. tenuis and C. flagellum and SSR markers for C. thwaitesii were identified. The diagnostic potential of these markers can be exploited to sex the Calamus species at the seedling stage for proper breeding and agroforestry management.

A sequence tagged site (STS) marker encoding Copia-like retrotransposable element is associated with male specific sex expression in Momordica dioica Roxb.

Momordica dioica (family—Cucurbitaceae), a perennial, dioecious climber with homomorphic sex chromosomes has been recently identified as a strategic plant for studying the early evolution of sex in angiosperms. Further, there is a greater demand for early sex identification in M. dioica because of its sex-specific medicinal and economic values. In this study, we used inter simple sequence repeat (ISSR) primers for developing a reliable molecular marker linked to sporophytic expression in M. dioica. Screening of bulked DNA pooled from male and female genotypes using 27 ISSR primers resulted in the detection of a male specific marker MdMSM. A sex-linked sequence tagged site (STS) marker MdY1 designed from MdMSM could efficiently differentiate the male and female M. dioica genotypes across different growth stages and geographical regions. Genome walking across the length of the sex-linked marker showed that MdY1 is highly homologous to the reverse transcriptase domain of a polyprotein and nested within a Copia-like retrotransposable element, MdRE1. Southern blot analysis revealed multiple copy locus and exclusive presence of MdY1 and MdRE1 only in the male genome suggesting that it might be exhibiting prominent Y-chromosome specific characteristics with strong expressions in the evolution of male sex organs. Thus, the STS marker MdY1 together with the Y-specific retroelement MdRE1 could be used as an efficient tool for early sex diagnosis in M. dioica, as well as a starting material towards characterization of traits involved in sex determination and evolutionary pathways of sexual dimorphism.

Gender Determination by Isolation of Cell-free Fetal DNA from the Maternal Circulation

ABSTRACT Objectives Early identification of fetal gender is important in management of X-linked and other metabolic disorders. Since ultrasound may not predict gender accurately during the first trimester, noninvasive fetal gender determination using deoxyribonucleic acid (DNA) amplification has been proposed. The aim of this study is to evaluate the feasibility of noninvasive prenatal gender determination by examining cell-free fetal DNA (cffDNA) from maternal plasma. Materials and methods Blood samples were collected from 49 pregnant women of gestational ages ranging from 12 to 41 weeks. Deoxyribonucleic acid was extracted from maternal plasma using a QIAamp DNA Blood Mini Kit. Real-time quantitative polymerase chain reaction (PCR) was performed to amplify the male specific DNA marker sex-determining region Y (SRY). Results From a total of 49 subjects, fetal gender was correctly determined in 13 out of 14 male fetuses and 32 out of 35 female fetuses, giving an overall accuracy of 92%. The sensitivity and specificity of the test to detect male fetuses was 93 and 91% respectively. There were three false-positive cases and one false-negative case. Conclusion Identification of fetal gender from maternal plasma using real-time PCR technique is feasible in a developing country, like Pakistan, and appears to be a promising tool for noninvasive prenatal diagnosis. How to cite this article Mohammed N, Nuruddin R, Hoodbhoy Z. Gender Determination by Isolation of Cell-free Fetal DNA from the Maternal Circulation. J South Asian Feder Obst Gynae 2016;8(1):29-32.

Identification of Sex-Specific Markers Reveals Male Heterogametic Sex Determination in Pseudobagrus ussuriensis.

Comprehending sex determination mechanism is a first step for developing sex control breeding biotechnologies in fish. Pseudobagrus ussuriensis, one of bagrid catfishes in Bagridae, had been observed to have about threefold size dimorphism between males and females, but its sex determination mechanism had been unknown. In this study, we firstly used the amplified fragment length polymorphism (AFLP)-based screening approach to isolate a male-specific DNA fragment and thereby identified a 10,569 bp of male-specific sequence and a 10,365 bp of female-related sequence by genome walking in the bagrid catfish, in which a substantial genetic differentiation with 96.35 % nucleotide identity was revealed between them. Subsequently, a high differentiating region of 650 bp with only 70.26 % nucleotide identity was found from the corresponding two sequences, and three primer pairs of male-specific marker, male and female-shared marker with different length products in male and female genomes, and female-related marker were designed. Significantly, when these markers were used to identify genetic sex of the bagrid catfish, only male individuals was detected to amplify the male-specific marker fragment, and female-related marker was discovered to produce dosage association in females and in males. Our current data provide significant genetic evidence that P. ussuriensis has heterogametic XY sex chromosomes in males and homogametic XX sex chromosomes in females. Therefore, sex determination mechanism of P. ussuriensis is male heterogametic XX/XY system.

Sex Identification in Polygamodioecious Simarouba (Simarouba glauca DC) Using RAPD and ISSR Markers

Simarouba (Simarouba glauca DC) is polygamodioecious perennial, popularized as an important oilseed tree crop for cultivation on wasteland. In simarouba only female plants are valued for commercial production of fruits and seeds, therefore early sex identification of male and female genotypes before planting becomes essential, as excess of male plants can be discarded at seedling stage. To distinguish male genotypes from female genotypes early in the seedling stage, 69 random amplified polymorphic DNA (RAPD) primers and 12 inter-simple sequence repeat (ISSR) primers were used to screen DNA bulk samples from 5 male and 15 female plants. Fifty three out of the 69 RAPD primers resulted in 373 reproducible RAPD bands with an average of 7 fragments amplified per primer. Five RAPD primers (OPC 06, OPC 16, OPD 15, OPE 05 and OPD 12) and two ISSR primers (ISSR 824 and ISSR 864) produced distinguishing banding pattern for male and female bulk samples. These seven markers were then used to screen individual 5 male and 15 female plants; RAPD primer OPE 05 consistently reproduced a male-specific fragment (521 bp), which was absent in all female plants. These results indicated that marker OPE-05521 may be a male-specific marker and may be used for sex characterization in simarouba at an early stage of plant growth.

Identification of male sex-linked DNA sequence of the cultured Pacific bluefin tuna Thunnus orientalis

A male-specific marker of a DNA sequence for the Pacific bluefin tuna Thunnus orientalis, Male delta 6 (Md6), was identified by comparative study of the sequences obtained by F3-cultured male and female AFLP-selective DNA amplification products, followed by high-throughput DNA sequencing. Md6 was characteristic in continuous 6-bp nucleotide deletions compared to redundant sequences that could be seen in both sexes. The PCR genotyping primers were developed based on the Md6 sequence, which showed that 29 of 32 F3 males (90.6 %) and 9 of 13 (69.2 %) F2 parental males were genotyping positive, whereas in females, the genotyping test was negative in parental F2 (n = 12), and 1 of 32 (3.1 %) F3 female was Md6-positive. Parentage allocation tests suggested that Md6-positive F2 males were more attendant in spawning than Md6-negative males, suggesting that Md6 was passed from male parents to male progeny under aquaculture conditions.

P04.26 * MOLECULAR MECHANISMS OF CIRCULATING MICRORNA BIOMARKERS FOR GLIOBLASTOMA

The development of biomarker panels for glioblastoma has the potential to greatly improve the outcome of treatment by assisting in earlier detection, determining response to treatment and predicting prognosis to improve therapeutic strategy. MicroRNAs (miRNAs) present in the circulation of glioblastoma patients provide a relatively non-invasive source of markers for this purpose. Gliomas, including glioblastoma, release miRNAs in exosomes and protein complexes which alter expression levels in the serum of patients. The aim of this study was to isolate and identify deregulated miRNAs from the sera of glioblastoma patients which could be used as biomarkers. Further to this, the molecular mechanisms behind the deregulation of these miRNA biomarkers were also determined. Total RNA was isolated from the sera of glioblastoma patients, patients undergoing elective surgery and volunteers, using phenol-chloroform extraction. Relative expression of miRNAs was determined using qRT-PCR and normalized using a synthetic spike in. Data was analysed using GraphPad Prism 5 software using the delta CT method. Statistical significance was determined using a t-test and one way ANOVA, a p-value of <0.05 was considered significant. Three miRNAs were significantly deregulated in the sera of glioblastoma patients compared to control sera. One prognostic marker determined by Kaplan-meier analysis, one male specific marker and one age-specific marker which could be used as a panel for glioblastoma patients. Further work using glioblastoma tissue will elucidate the role of these miRNAs in glioblastoma including the underlying molecular differences between patients exhibiting deregulated miRNA and subsequently improved prognosis.