Endogenous Reference Genes Research Articles

Identification of miR-328-3p as an endogenous reference gene for the normalization of miRNA expression data from patients with Diabetic Retinopathy

Diabetic Retinopathy, the main cause of visual loss and blindness among working population, is a complication of Diabetes mellitus (DM), which has been described as a major public health challenge, so it is important to identify biomarkers to predict and to stratify patient´s possibility for developing DR. MicroRNAs (miRNAs) are small non-coding RNA molecules that have showed to be promising disease biomarkers and association of miRNAs with the possibility to develop DR has been reported. However, evaluating miRNA expression involves normalization of RT-qPCR data using internal reference genes that should be properly determined, considering their impact on expression levels calculation and, until date, there is no unanimity on reference miRNAs for the investigation of circulating miRNAs in DR. We aimed to estimate the appropriateness of a group of miRNAs as normalizers to identify which might be considered steady internal reference genes in expression studies on DR plasma samples. Expression levels of candidates were analyzed in 60 healthy controls, 48 DM without DR patients and 62 DR patients with two statistical tools: NormFinder and RefFinder. MiR-328-3p was the most stable gene and we also investigated the effect of gene normalization, demonstrating that different normalization strategies have important implications for accurate data interpretation.

Species-specific TM-LAMP and Trident-like lateral flow biosensor for on-site authenticity detection of horse and donkey meat

Global food research is placing increasing emphasis on the on- site authenticity detection of meat since it is vital for this process to be conducted accurately and efficiently. In this study, a trident-like lateral flow biosensor (LFB) for multiple on-site authenticity detection of horse and donkey meat was established for the first time based on species-specific TM-LAMP (tag-labeled multiplex loop-mediated isothermal amplification). Firstly, a novel, accurate model for screening chromosomal endogenous reference genes was constructed using Perl as the bioinformatics language, based on which the single-copy genes LOC106782588 and LOC106825524 were selected as the endogenous reference genes for horses and donkeys, respectively. Secondly, antigen tag-labeled multiplex LAMP based lateral flow biosensor was established. Following optimization, the limit of detection (LOD) reached a level as low as 40 pg, which was equivalent to 15 copies in horses and donkeys. TM-LAMP was proved to be suitable for on-site detection since the process took only 40 min, with no need of any precision instruments. The analysis of various processed foods and mixed meat products indicated that this biosensor displayed high specificity and sensitivity consistently. As a universal platform, this biosensor can be custom-designed for on-site detection of other species by changing specific primers, which might be an excellent prospect for the identification of species authenticity.

Verification and applicability of endogenous reference genes for quantifying GM rice by digital PCR

To standardize the rice-specific quantification methods, the criteria of six genes of rice (gos9, PLD, SPS, RBE4, ppi-PPF and oriazain) were compared and evaluated by ddPCR. The results revealed that SPS, RBE4 and ppi-PPF were single copy genes per haploid genome and species specificity and stable among different rice cultivars, by employing Lectin gene of soybean as internal reference gene. The established ddPCR systems were precise and reliable with an absolute LOQ of 10–20 copies/reaction. Furthermore, the robustness of these three assays was verified by performing an intra-laboratory repeatability validation and the results showed that the three endogenous genes of rice could be quantitated repeatedly and precisely above the LOQ. These ddPCR methods can reliably quantified the GM content even if the content was low to 0.1%, which were much more reliable than the results from real-time PCR using the same primers and probes.

The quantitative determination method of covalently closed circular DNA HBV in puncture biopsy specimens of the liver.

To analyze the method HBV covalent-closed circular DNA quantitative determination in liver puncture biopsies and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of liver tissue biopsy material were used from 128 patients living in St. Petersburg, in various regions of the Russian Federation, as well as in the Republic of Uzbekistan. For quantitative analysis of HBV covalently closed circular DNA in a biopsy material a method was developed based on real-time PCR using TaqMan probes for the target fragment and for the endogenous reference gene, based on the detecting ccc HBV DNA method of Pollicino T. et al. When quantifying ccc DNA HBV in liver tissue of 18 moderately HBV activity with HBV DNA PCR positive results patients and 16 inactive HBsAg carriers, the ccc DNA HBV content was significantly different between groups (p<0.034) and in terms 1 copy of the β-globin gene among moderate activity HBV patients amounted to 1.71±1.32 copies/cell, and for inactive HBsAg carriers 0.15±0.14 copies/cell. In the group of patients with severe liver fibrosis and cirrhosis, the amount of ccc DNA HBV in liver tissue in patients with HBV averaged 2.5±0.4 copies/cell, in patients with HBV + D on average 0.7±0.25 copies/cell, in patients with HCV + HBV co-infection 0.45±0.07 copies/cell, in patients with a preliminary diagnosis of chronic hepatitis C hepatitis, on average 0.12±0.04 copies/cell, in patients with cryptogenic hepatitis 0.2± 0.05 copies/cell. A significant difference was shown between the group of patients with chronic hepatitis B with marked fibrosis and cirrhosis of the liver with other patients groups, except for the group of 18 moderate activity chronic hepatitis B patients. The values of Student's t-test when compared with other groups were respectively: for patients with a HCV preliminary diagnosis t=5,92 p<0,05 f = 19, patients with cryptogenic hepatitis t=5,71 p<0,05 f = 18, with «inactive HBsAg carriage» t=5,55 p<0,05 f = 29, with HCV + HBV co-infection t=5,05 p<0,05 f = 15 and HBV + D co-infection t=3,82 p<0,05 f = 17. The covalently closed circular DNA HBV quantitative assessment method in liver puncture biopsies allows identifying HBsAgnegative chronic viral hepatitis B forms and also reflects the virus replication activity, which, in turn, makes it possible to assume further disease progression and evaluate the antiviral therapy effectiveness.

Human Circulating miRNAs Real-time qRT-PCR-based Analysis: An Overview of Endogenous Reference Genes Used for Data Normalization.

miRNAs are small non-coding RNAs of about 18–25 nucleotides that negatively regulate gene expression at the post-transcriptional level. It was reported that a deregulation of their expression patterns correlates to the onset and progression of various diseases. Recently, these molecules have been identified in a great plethora of biological fluids, and have also been proposed as potential diagnostic and prognostic biomarkers. Actually, real time quantitative polymerase chain reaction is the most widely used approach for circulating miRNAs (c-miRNAs) expression profiling. Nevertheless, the debate on the choice of the most suitable endogenous reference genes for c-miRNAs expression levels normalization is still open. In this regard, numerous research groups are focusing their efforts upon identifying specific, highly stable, endogenous c-mRNAs. The aim of this review is to provide an overview on the reference genes currently used in the study of various pathologies, offering to researchers the opportunity to select the appropriate molecules for c-miRNA levels normalization, when their choosing is based upon literature data.

Analysis of RNA in the estimation of post-mortem interval: a review of current evidence.

Post-mortem interval (PMI) determination is one of the most important issues in forensic sciences. In the past, forensic scientists provided different approaches (physical, chemical, and entomological) for the estimation of PMI without success.However, advances in molecular biology over the last two decades have allowed us to assess the time-dependent degradation of biological markers (e.g., proteins, DNA, and RNA). Thus, the aim of the manuscript is to provide a review of the recent progress in the estimation of PMI using molecular biology methods, mainly focusing on the potential usefulness of RNA markers. To this end, 29 studies have been systematically reviewed, each one chosen according to specific inclusion/exclusion criteria. The selected studies evaluated the contribution of endogenous reference genes in different biological samples in order to determine the PMI based on post-mortem RNA degradation as a function of other influencing factors such as time, cause of death, and environmental conditions.

Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

Validation of endogenous reference genes in rat cerebral cortex for RT-qPCR analyses in developmental toxicity studies

Relative gene expression data obtained from quantitative RT-PCR (RT-qPCR) experiments are dependent on appropriate normalization to represent true values. It is common to use constitutively expressed endogenous reference genes (RGs) for normalization, but for this strategy to be valid the RGs must be stably expressed across all the tested samples. Here, we have tested 10 common RGs for their expression stability in cerebral cortex from young rats after in utero exposure to thyroid hormone (TH) disrupting compounds. We found that all 10 RGs were stable according to the three algorithms geNorm, NormFinder and BestKeeper. The downstream target gene Pvalb was significantly downregulated in brains from young rats after in utero exposure to propylthiouracil (PTU), a medicinal drug inhibiting TH synthesis. Similar results were obtained regardless of which of the 10 RGs was used for normalization. Another potential gene affected by developmental TH disruption, Dio2, was either not affected, or significantly upregulated about 1.4-fold, depending on which RG was used for normalization. This highlights the importance of carefully selecting correct RGs for normalization and to take into account the sensitivity of the RT-qPCR method when reporting on changes to gene expression that are less than 1.5-fold. For future studies examining relative gene expression in rat cerebral cortex under toxicological conditions, we recommend using a combination of either Rps18/Rpl13a or Rps18/Ubc for normalization, but also continuously monitor any potential regulation of the RGs themselves following alterations to study protocols.

Selection of suitable reference genes for core clock gene expression analysis by real-time qPCR in rat ovary granulosa cells.

Selection of a suitable endogenous reference gene is essential for investigating expression of clock genes Bmal1, Clock, Pers, Crys, Rev-erbα/β, and RORα/β/γ involved in the circadian system. In this study, we treated rat ovary granulosa cells with dexamethasone to synchronize circadian oscillation in vitro and determined expression levels of Bmal1 and Per2 and six candidate reference genes (Actb, Beta actin; B2m, Beta-2-microglobulin; Ppia, Cyclophilin A; Gapdh, Glyceraldehyde-3-phosphate dehydrogenase; Hprt, Hypoxanthine guanine phosphoribosyl transferase and Tbp, TATA-box-binding protein) using quantitative real-time PCR. We then employed three software programs, GeNorm, NormFinder, and BestKeeper, to analyze the expression data for the selection of the best reference gene. According to GeNorm, Tbp and B2m were assessed as the most stable reference genes; Tbp and Hprt were best by NormFinder and BestKeeper, respectively. Thus, we recommend Tbp as the most suitable reference gene for studying clock genes expression in rat ovary granulosa cells in vitro.

Identification of suitable reference microRNA for qPCR analysis in pediatric inflammatory bowel disease.

Pediatric inflammatory bowel disease (IBD) accounts for 10-15% of IBD and is associated with considerable morbidity for patients. Dysregulated microRNAs (miRNA, miR), small noncoding RNA molecules that modulate gene expression, have been the target of research in IBD diagnosis, surveillance, and therapy. Proper selection of reference genes, which are a prerequisite for accurate measurement of miRNA expression, is currently lacking. We hypothesize that appropriate normalization requires unique reference genes for different tissue and disease types. Through the study of 28 pediatric intestinal samples, we sought to create a protocol for selection of suitable endogenous reference genes. Candidate reference genes (miR-16, 193a, 27a, 103a, 191) were analyzed by RT-quantitative (q)PCR. Criteria used for designation of suitable reference genes were as follows: 1) ubiquitous: present in all tissue samples with quantification cycle value 15-35; 2) uniform expression: no differential expression between control and disease samples (P > 0.05); 3) stability: stability value <0.5 by NormFinder. Our results suggest the use of miR-27a/191 for Crohn's disease small bowel, none of the five candidate genes for Crohn's disease colon, and miR-16/27a for ulcerative colitis. Additionally, target miR-874 had differential expression when normalized with different reference genes. Our results demonstrate that reference gene choice for qPCR analysis has a significant effect on study results and that proper data normalization is imperative.

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation.

Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs.

Selection of endogenous reference genes for qRT-PCR analysis in Camelina sativa and identification of FLOWERING LOCUS C allele-specific markers to differentiate summer- and winter-biotypes

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis greatly relies on transcript normalization using stably expressed reference genes. This study identifies reference genes for qRT-PCR analysis in different organs and vernalized tissues of camelina (Camelina sativa). Nineteen stably expressed candidate reference genes from 22,157 genes were identified by RNAseq analysis of pre- and post-vernalized tissues of a summer- (CO46) and winter- (Joelle) biotype. Two additional candidate reference genes were also selected from orthologs of arabidopsis encoding ACTIN2 (ACT2) and NOT INDUCED BY SCLEROTINIA INFECTION (NIS). We also evaluated the transcript levels of three camelina plant genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), FLOWERING LOCUS C (FLC), and MADS AFFECTING FLOWERING 2 (MAF2), which are known to be differentially-regulated in response to cold temperatures. The stability of transcript abundance was ranked using NormFinder, geNorm, BestKeeper, and Comparative ΔCT software. EXOCYST COMPLEX COMPONENT SEC3A (SEC3A), UBIQUINONE OXIDOREDUCTASE (UbOxRed), and RING-U-BOX domains-containing protein (RUB) were the most suitable reference genes for normalization of gene expression in camelina. In this study, SEC3A was used to normalize qRT-PCR expression data obtained using two sets of allele-specific FLC markers to differentiate summer- and winter-biotypes across 30 accessions of camelina.

Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification

Cisgenic plants must be free of exogenous genetic elements such as antibiotic or herbicide resistance genes commonly used in the selection phase of a gene transfer protocol. However, the use of a selection marker is essential for the transformation of many fruit crops, including apple (Malus × domestica), for which the efficiency of DNA integration is very low. Currently, the approach with the highest chances of success relies on the removal of undesired exogenous genes by means of an inducible site-specific recombinase enzyme and its recognition sites. We developed a quantitative, rapid and cost-effective method based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. MdTOPO6 gene was chosen as endogenous reference gene for apple due to the single-copy presence in the haploid genome and to the species-specificity. A recombinant plasmid harboring specific sequences of both the reference gene and the target gene nptII was used as calibrator to build the standard curves. The limit of quantification of the method was evaluated, and precision and trueness of the quantification performances proved to be valid according to international reference criteria. Finally, this method was applied to characterize transgenic and cisgenic apple lines, and to investigate the possibility of removing an entire T-DNA cassette from the genome of edited apple plants.

Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification

Cisgenic plants must be free of exogenous genetic elements such as antibiotic or herbicide resistance genes commonly used in the selection phase of a gene transfer protocol. However, the use of a selection marker is essential for the transformation of many fruit crops, including apple (Malus × domestica), for which the efficiency of DNA integration is very low. Currently, the approach with the highest chances of success relies on the removal of undesired exogenous genes by means of an inducible site-specific recombinase enzyme and its recognition sites. We developed a quantitative, rapid and cost-effective method based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. MdTOPO6 gene was chosen as endogenous reference gene for apple due to the single-copy presence in the haploid genome and to the species-specificity. A recombinant plasmid harboring specific sequences of both the reference gene and the target gene nptII was used as calibrator to build the standard curves. The limit of quantification of the method was evaluated, and precision and trueness of the quantification performances proved to be valid according to international reference criteria. Finally, this method was applied to characterize transgenic and cisgenic apple lines, and to investigate the possibility of removing an entire T-DNA cassette from the genome of edited apple plants.

Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

This protocol describes a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using a two-enzyme, two-tube approach, carried out using either SYBR Green I or TaqMan chemistries. The protocol uses a PCR volume of 20 µL (although most manufacturers recommend 50-µL reactions). However, if the PCR target is not very abundant (i.e., present at one to 10 copies per sample), a larger volume may yield better reproducibility between samples. Discussion on preparing high-quality RNA, choosing a priming method, selecting an enzyme, and selecting an endogenous reference gene is also included.

Loop-Mediated Isothermal Amplification for Detection of Endogenous Sad1 Gene in Cotton: An Internal Control for Rapid Onsite GMO Testing.

Confirming the integrity of seed samples in powdered form is important prior to conducting a genetically modified organism (GMO) test. Rapid onsite methods may provide a technological solution to check for genetically modified (GM) events at ports of entry. In India, Bt cotton is the commercialized GM crop with four approved GM events; however, 59 GM events have been approved globally. GMO screening is required to test for authorized GM events. The identity and amplifiability of test samples could be ensured first by employing endogenous genes as an internal control. A rapid onsite detection method was developed for an endogenous reference gene, stearoyl acyl carrier protein desaturase (Sad1) of cotton, employing visual and real-time loop-mediated isothermal amplification (LAMP). The assays were performed at a constant temperature of 63°C for 30min for visual LAMP and 62ºC for 40min for real-time LAMP. Positive amplification was visualized as a change in color from orange to green on addition of SYBR® Green or detected as real-time amplification curves. Specificity of LAMP assays was confirmed using a set of 10 samples. LOD for visual LAMP was up to 0.1%, detecting 40 target copies, and for real-time LAMP up to 0.05%, detecting 20 target copies. The developed methods could be utilized to confirm the integrity of seed powder prior to conducting a GMO test for specific GM events of cotton. LAMP assays for the endogenous Sad1 gene of cotton have been developed to be used as an internal control for onsite GMO testing in cotton.

Acetolactate Synthase Overexpression in Mesosulfuron-Methyl-Resistant Shortawn Foxtail ( Alopecurus aequalis Sobol.): Reference Gene Selection and Herbicide Target Gene Expression Analysis.

Severe infestations of shortawn foxtail ( Alopecurus aequalis Sobol.), a noxious weed in wheat and canola cropping systems in China, remain standing even after the application of the herbicides, fenoxaprop- P-ethyl and mesosulfuron-methyl. Analysis of gene expression in weed plants subjected to herbicide treatment is a key step toward more mechanistic studies. Since such an analysis often involves quantitative real-time PCR (qRT-PCR), endogenous reference genes with stable expression are required. Herein, we identified specific gene sets, suitable as references for qRT-PCR data normalization in A. aequalis plants under different experimental conditions, using geNorm, NormFinder, BestKeeper, and RefFinder software. Additionally, the reliability of reference genes was verified by analyzing the expression of genes encoding two major herbicide target enzymes: acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS). Furthermore, functional ALS gene amplification was likely present in resistant plants, although it may make no contribution to the resistant phenotypes.

Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate the expression of genes, and they affect important biological and physiological states. Circulating miRNAs in blood are useful markers of metabolism and economic traits. Expression levels of circulating miRNAs have been estimated using quantitative real-time PCR (qPCR). Proper normalization is critical for accurate miRNA expression analysis. However, there is no study which systematically presented endogenous reference genes for evaluating circulating miRNA expression in pigs. In this study, ten porcine miRNAs (let-7a, miR-16, miR-17, miR-23a, miR-26a, miR-93, miR-103, miR-107, miR-127 and miR-191), based on the literature, were chosen as candidate reference miRNAs in serum. We evaluated the expression stability value of these miRNAs in Berkshire, Duroc, Landrace and Yorkshire pigs using geNorm and NormFinder. We determined the optimal combination of reference miRNAs for qPCR experiments: miR-127 and miR-17 in Berkshire pigs; miR-127 and miR-93 in Duroc and Landrace pigs; miR-127 and miR-16 in Yorkshire pigs. miR-127 was the best reference gene in pigs, regardless of the breed. Our study is crucial for the discovery of novel biomarkers in pigs. The reference miRNAs presented in this study could be used as appropriate reference genes for the measurement of circulating miRNA levels in studies of physiological blood metabolites.

Expressed repetitive elements are broadly applicable reference targets for normalization of reverse transcription-qPCR data in mice

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data.

Diagnostic utility of glycosyltransferase mRNA expression in gastric cancer

Objective/backgroundPosttranslational modification of proteins, including glycosylation, is known to differ between normal and tumor cells. Altered glycosyltransferase levels have been observed in tumor tissues and their role in tumor metastasis and invasion has been implicated. In this study the role of altered glycosyltransferase messenger RNA (mRNA) levels in serum of gastric cancer patients as early markers of gastric cancer was evaluated. MethodsIn this case control study the expression profile of ppGalNAc-T6, GlcNAcT-V, ST3Gal I, ST3 Gal IV, and ST6GalNAc-I in normal healthy control and gastric cancer patients was compared. Serum was isolated from blood samples of gastric cancer patients (n = 200) and controls (n = 200). Following RNA extraction, reverse transcription was carried out and transcript levels of glycosyltransferases were determined using real-time quantitative polymerase chain reaction and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The amount of target gene, normalized to an endogenous reference gene relative to calibrator was calculated by using ΔΔCT method. Transcript levels in the serum samples of gastric cancer patients were compared with those of controls; also the same was correlated within sex and different stages of disease. ResultsThe mRNA expression of ppGalNAc-T6 and ST6GalNAc-I was significantly higher in serum samples of gastric cancer patients on comparison with controls (p = .008), however, there was no significant difference in mRNA expression of GlcNAcT-V, ST3Gal I, and ST3 Gal IV in serum samples of gastric cancer patients and controls (p = .097). In addition, no significant association of mRNA expression of these glycosyltransferases was found within sex and stages in this study. ConclusionThis study revealed the potential of ppGalNAc-T6 and ST6GalNAc-I mRNA transcript levels in serum as markers of gastric cancer. Further studies on the wider range of glycosyltransferases in various cancers are needed to establish signature mRNA batteries as minimally invasive markers of gastric cancer.