Endogenous Reference Research Articles

Analysis of circulating microRNA during early gestation in Japanese black cattle

Circulating microRNAs (miRNAs) have been used as biomarkers for various diseases and physiological conditions in humans and mice; studies in domestic animals, particularly cattle, are limited. The importance of early pregnancy diagnosis (especially within the 21-d cow estrous cycle) in the livestock industry is extremely high. This study compared the circulating miRNAs in bred non-pregnant and pregnant Japanese Black cows, explored miRNAs as biomarkers for early pregnancy diagnosis, and established a measurement system that included selecting an appropriate reference miRNA and determining the effect of hemolysis on miRNA quantification in plasma. miRNA was extracted from the plasma of Japanese Black cows on day 21 after artificial insemination and subjected to a customized bovine oligonucleotide microarray for expression analysis. Differentially expressed miRNAs and reference miRNA candidates were selected and validated using reverse transcription-quantitative PCR (RT-qPCR). An appropriate endogenous reference miRNA for normalization was selected using NormFinder software. To evaluate the effect of hemolysis on miRNA quantification, hemolyzed samples were prepared using plasma from four cows in the estrous cycle and subjected to RT-qPCR. A total of 124 miRNAs were detected in bovine plasma by microarray analysis in bred non-pregnant and pregnant cows. The levels of five circulating miRNAs were significantly higher in pregnant cows than in bred non-pregnant cows, and 24 miRNAs were detected only in the pregnant group. NormFinder analysis and RT-qPCR validation showed that miR-2455 was an appropriate reference miRNA in the plasma of bred non-pregnant and pregnant Japanese Black cows, and miR-19b, miR-25, miR-29a, and miR-148a were significantly higher in the pregnant group. These four circulating miRNAs did not change during the estrous cycle and were less affected by hemolysis. In the current study, we found four miRNAs, miR-19b, miR-25, miR-29a, and miR-148a, which were present at high levels in the plasma of pregnant Japanese Black cows. Since these miRNAs are less affected by hemolysis, they may potentially be used as biomarkers for early pregnancy diagnosis in cattle.

Importance of miR-141-5p and miR-501-5P expression in patients with HBV infection.

MicroRNAs (miRNAs) as small RNA and post-transcriptional modulators are shown to have regulatory effects for different cellular activities and pathways, such as metabolism, virus replication and also cell growth. In addition, miRNAs can regulate the replication of the hepatitis B virus (HBV). Therefore, the expression profile of miRNAs was evaluated in HBV-infected patient groups and healthy controls. The expression levels of the following microRNAs (as noninvasive biomarkers) were compared in two experimental (those with various stages of HBV infection) and control groups to evaluate their diagnosis ability: mir141-5p and mir501-5p. RNA extraction was performed for 45 serum samples. The miRCURY LNA™ Universal RT-miRNA-PCR system and miRNA PCR panels were used for measuring microRNA expression profiles. To normalize quantitative values, the endogenous reference by UniSp6 expression was used. Serum mir141-5p and mir501-5 were significant None in patient in different stages of HBV infection(p<0.001) than in controls(p<0.01). Receiver operating characteristic (ROC) curve analyses suggested that serum has mir141-5p and mir501-5p none significant diagnostic value for HBV infection. Results suggest that mir141-5p and mir501-5 can not be used as diagnostic biomarkers for monitoring of HBV infection and other biomarkers in this disease need to be investigated.

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models

The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies.

High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis.

It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene hmg was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed hmg gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of bar and hmg with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the bar and hmg genes varied from 1.18 to 2.94%. The copy numbers of the transgene bar using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T1 and T2 progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new hmg gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.

Identification of Gene Copy Number in the Transgenic Plants by Quantitative Polymerase Chain Reaction (qPCR).

Transgenic events are defined as exogenous DNA insertion in the genome through genetic transformation. It is a powerful means for the improvement of crop plants and to understand the gene function. Multiple DNA insertion events may occur at one or several chromosomal locations. One of the important tasks, after validation of the transformation of transgenic plants, is the identification of single copy inthe transgenic. This means the insertion of exogenous DNA fragment only in a single locus in the genome. Southern blot hybridization is a convincing and reliable method, for estimation of copy number in transgenic lines but it is cumbersome and time-consumingprocess. One of the other well-known methods is quantitative polymerase chain reactions (qPCR), a simple and rapid method to identify copy number from a population of independent transgenic lines. In comparison to the Southern hybridization method, qPCR is simpler to perform, requires less DNA, lesser time and does not require any labeled probes. This method utilizes specific primers to amplify target transgenes and endogenous reference genes. Designing an appropriate and specific primer pair is a very crucial part of the estimation of the gene copy number. In this chapter, we have illustrated a detailed methodology for identification of the gene copy of the transgenic plants.

Circulating Exosomal miRNAs as Biomarkers in Epithelial Ovarian Cancer.

Failure to detect early-stage epithelial ovarian cancer (EOC) is a major contributing factor to its low survival rate. Increasing evidence suggests that different subtypes of EOC may behave as distinct diseases due to their different cells of origins, histology and treatment responses. Therefore, the identification of EOC subtype-specific biomarkers that can early detect the disease should be clinically beneficial. Exosomes are extracellular vesicles secreted by different types of cells and carry biological molecules, which play important roles in cell-cell communication and regulation of various biological processes. Multiple studies have proposed that exosomal miRNAs present in the circulation are good biomarkers for non-invasive early detection of cancer. In this review, the potential use of exosomal miRNAs as early detection biomarkers for EOCs and their accuracy are discussed. We also review the differential expression of circulating exosomal miRNAs and cell-free miRNAs between different biofluid sources, i.e., plasma and serum, and touch on the issue of endogenous reference miRNA selection. Additionally, the current clinical trials using miRNAs for detecting EOCs are summarized. In conclusion, circulating exosomal miRNAs as the non-invasive biomarkers have a high potential for early detection of EOC and its subtypes, and are likely to be clinically important in the future.

235 Uterine Function During Maternal Recognition of Pregnancy Differs Due to Size of the Ovarian Reserve in Beef Heifers

Abstract The relationship of ovarian reserve parameters to fertility in mammalian females continues to be debated. We demonstrated improved uterine function, creating an environment that is more supportive of early embryonic development in beef heifers with increased numbers of follicles. In the present study we hypothesized that timing of luteolysis differs between heifers with increased compared to heifers with diminished numbers of follicles. Angus heifers (n = 20/ group) were classified as low (14.7 ± 1.1) or high (32 ± 1.1) antral follicle number based on ovarian ultrasonography, and artificially inseminated (d0) following a Select Synch protocol. At d16 after insemination, heifers were slaughtered, and reproductive tracts collected. Tracts were flushed with 20 mL of physiological saline to determine pregnancy status by presence of a conceptus and endometrial samples from the horn ipsilateral to the CL were frozen for determination of transcript abundance. Real-time RT-PCR was performed to determine relative transcript abundance of oxytocin receptor (OXTR) and interferon-stimulated gene-15 (ISG15) with glyceraldehyde-3-phospate dehydrogenase as the endogenous reference gene. Transcript abundance was analyzed using the MIXED procedure of SAS with follicle group, pregnancy status and the interaction as fixed effects. Transcript abundance of ISG15 was greater in pregnant endometrium than non-pregnant endometrium (2.75 ± 0.25 vs 0.16 ± 0.22-fold; P &amp;lt; 0.0001) with no influence of follicle group or the interaction. The interaction of follicle group and pregnancy status influenced OXTR transcript abundance (P = 0.06). Luteolytic mechanisms were activated in non-pregnant heifers with diminished numbers of follicles (1.46 ± 0.25-fold) and were not activated in pregnant heifers (0.06 ± 0.22-fold) or heifers with increased numbers of follicles that were not pregnant (0.32 ± 0.30-fold). These data indicate a wider window of recognition of pregnancy in beef heifers with increased numbers of ovarian follicles. USDA is an equal opportunity provider and employer.

Selection and validation of endogenous reference genes for RT-qPCR normalization in different stresses and tissues of the tiger milk mushroom, Pleurotus tuber-regium.

Pleurotus tuber-regium is a sclerotium-producing mushroom with pharmacological and nutritional value. Many researches applied molecular biological strategies that allow analyzing numerous genes expressed in response to different growth stresses or tissues. In order to capture accurate and reliable results in gene expression studies, it is necessary to select appropriate internal reference genes. They must have relatively uniform expression in the majority of tested samples, thereby reducing the impact of the sample itself on the RT-qPCR results. The selection and validation of 14 candidate reference genes, including α-tub, β-tub, γ-tub, GAPDH, Tif-5a, Tef-1α, ATPase, acyl-CoA, U2ribo, S/G, Wlp, VtpAsE, E3upl, and Sdh, were carried out for gene expression analysis in P. tuber-regium mycelia in response to different temperatures, drought levels, and salinity shifts, fruitbody, and sclerotium. Four statistical algorithms, NormFinder, geNorm, BestKeeper, and RefFinder, were recruited to evaluate the transcription stability of candidate reference genes. The RT-qPCR expression stability analysis indicated that the E3upl and Tif-5a were the most stable expressed genes among all experimental samples, so the combination of these two reference genes is suitable for the analysis of gene expression patterns in P. tuber-regium.

5α-reductase inhibitors: Evaluation of their potential confounding effect on GC-C-IRMS doping analysis.

5α-reductase inhibitors (5-ARIs) are considered by the World Anti-doping Agency as potential confounding factors in evaluating the athlete steroid profile, since they may interfere with the urinary excretion of several diagnostic compounds. We herein investigated 5α-reductase inhibitors from a different perspective, by verifying their influence on the carbon isotopic composition of 5α- and 5β-reduced testosterone and nandrolone metabolites. The GC-C-IRMS analysis was performed on a set of urine samples collected from three male Caucasian volunteers after the acute and chronic administration of finasteride in combination with the intake of 19-norandrostenedione, a nandrolone precursor. The excretion and the isotopic profile of androsterone (A), etiocholanolone (Etio) 5α-androstane-3α,17β-diol (5αAdiol), and 5β-androstane-3α,17β-diol (5βAdiol) were determined as well as those of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Pregnanediol (PD) and pregnanetriol (PT) were also measured as endogenous reference compounds to define the individual endogenous isotopic profile. Our results confirmed the impact of finasteride, especially if chronically administered, on the enzymatic pathway of testosterone and nandrolone, and pointed out the influence of 5-ARIs on δ13 C values of the selected target compounds determined in the IRMS confirmation analysis.

Who Is High Income, Anyway? Social Comparison, Subjective Group Identification, and Preferences over Progressive Taxation

Why are high-income and low-income earners not significantly polarized in their support for progressive income taxation? This article posits that the affluent fail to recognize that they belong to the high-income income group and this misperception affects their preferences over progressive taxation. To explain this mechanism theoretically, I introduce a formal model of subjective income-group identification through self-comparison to an endogenous reference group. In making decisions about optimal tax rates, individuals then use these subjective evaluations of their own income group and earnings of other groups. Relying on ISSP data, I find strong evidence for the model’s empirical implications: most high-income earners support progressive taxation when they identify themselves with a lower group. Additionally, individuals who overestimate the earnings of the rich are more likely to support progressive taxation.

Carbon isotope ratios of endogenous steroids found in human serum\u2014method development, validation, and reference population-derived thresholds

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)–based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples. In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated taking into account the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes. The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T replacement therapies and excretion study samples were investigated.Graphical abstract

Identification of Tps2 Used as an Endogenous Reference Gene in Qualitative and Real-time Quantitative PCR Detection of Flammulina velutipes

Identification of Tps2 Used as an Endogenous Reference Gene in Qualitative and Real-time Quantitative PCR Detection of Flammulina velutipes

Improving the cost-effectiveness of the Conservation Reserve Program: A laboratory study

The Conservation Reserve Program (CRP) is arguably the world's largest payments-for-ecosystem services program, with $1.8 billion paid to farmers in 2017 for practices on 23.4 million acres. The CRP uses a pay-as-bid, reverse auction with field-specific price caps to enroll most of the land in the program. Economic theory and empirical studies from other domains suggest that the restrictive price-cap auction format used in the current design of the CRP exhibits issues in terms of efficiency and cost-effectiveness. Using a laboratory experiment, we study the impact of varying the tightness of the price-cap auctions. We also examine two alternative auction formats based on reference prices. We find that excessively tight bid caps reduce efficiency and cost effectiveness by discouraging participation. Conversely, bid caps set too high also reduce cost-effectiveness by allowing higher rents. An exogenous reference price ranking format, which makes medium-cost sellers more competitive against low-cost sellers, reduces both efficiency and cost-effectiveness. An endogenous reference price format increases cost-effectiveness by increasing participation and reducing rents.

Identification of optimal and novel reference genes for quantitative real‐time polymerase chain reaction analysis in grapevine

Background and Aims Quantitative real-time polymerase chain reaction (qRT-PCR) has the advantages of sensitivity, specificity and repeatability, and is commonly used to investigate the expression levels of genes. The selection of appropriate endogenous reference genes is the pre-condition for acquiring the desired results of qRT-PCR. This study aimed to identify and screen the most suitable reference genes for grapevines. Methods and Results Three novel genes from previous transcriptomic data were selected for their stable expression either at the developmental stages of the grape berry or under the various treatments. The expression stability of these three candidate genes and six commonly used reference genes were evaluated with geNorm, NormFinder and BestKeeper algorithms in several grapevine samples. The geNorm analysis indicated that two reference genes were sufficient to normalise expression. The optimal reference gene combinations were: VvEF1-γ and PPR2 in fruit; RRM1 and EF1-α in leaf; EF1-α and Actin in tendril of several cultivars. The combination of EF1-α and VvEF1-α was the most appropriate reference gene for fruits at different developmental stages. When all the samples were considered, the combination of RRM1 and Ubiquitin was optimal. Conclusions Different reference genes should be employed when qRT-PCR is performed in different grapevine samples. The gene RRM1 is a new appropriate reference gene for grapevine. Significance of the Study This study provided alternatives for the optimal reference genes in qRT-PCR, and identified one novel and appropriate reference gene (RRM1) for grapevine.

Candidate reference genes for quantitative gene expression analysis in Lagerstroemia indica.

Quantitative gene expression analysis by qPCR requires reference genes for normalization. Lagerstroemia indica (crape myrtle) is a popular ornamental plant in the world, but suitable endogenous reference genes are lacking. To find suitable reference genes, we evaluated the stabilities of nine candidate genes in six experimental data sets: six different tissues, three leaf colors, nine flower colors, and under three abiotic stresses (salt, drought, cold) using four statistical algorithms. A target gene LiMYB56 (homolog of Arabidopsis MYB56) was used to verify the authenticity and accuracy of the candidate reference genes. The results showed that the combination of two stably expressed reference genes, rather than a single reference gene, improved the accuracy of the qPCR. LiEF1α-2 + LiEF1α-3 was best for the tissue, salt treatment, and drought treatment sets; LiEF1α-2 + LiEF1α-1 was optimal for leaf color; LiEF1α-2 + LiACT7 was optimal for cold treatment; and LiUBC + LiEF1α-1 was best for the flower color set. Notably, LiEF1α-2 had high expression stability in all six experimental sets, implying it may be a good reference gene for expression studies in L. indica. Our results will facilitate future gene expression studies in L. indica.

Carrying out pseudo dual nucleic acid detection from sample to visual result in a polypropylene bag with CRISPR/Cas12a

Amplification-based nucleic acid detection is widely employed in food safety, medical diagnosis and environment monitoring. However, conventional nucleic acid analysis has to be carried out in laboratories because of requiring expensive instruments and trained personnel. If people could do nucleic acid detection at home by themselves, the application of nucleic acid detection would be greatly accelerated. We herein reported a polypropylene (PP) bag-based method for convenient detection of nucleic acids in the oil-sealed space. The PP bag has three chambers which are responsible for lysis, washing and amplification/detection, respectively. After adding sample, nucleic acids are adsorbed on magnetic particles (MPs) and moved into these three chambers successively through immiscible oil channel by an external magnet. Combined with isothermal amplification, the PP bag can be incubated in a water bath or milk warmer and acted as a reaction tube. With highly specific CRISPR technology, Salmonella typhimurium (St) and SARS-CoV-2 can be visually detected in these PP bags within 1 h, indicating its potential household application. To further improve the reliability of nucleic acid testing at home, a logic decision method is introduced by detecting both target and endogenous reference gene. Positive/negative/invalid detection result can be obtained by chronologically adding the CRISPR reagents of target and endogenous reference gene. We anticipate that this PP bag can provide a novel toolkit for nucleic acid detection in people's daily life.

Detecting the abuse of 19-norsteroids in doping controls: A new gas chromatography coupled to isotope ratio mass spectrometry method for the analysis of 19-norandrosterone and 19-noretiocholanolone.

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with β-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ13 C values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of "unstable" urine samples, in late excretion phases or when coadministration with 5α-reductase inhibitors occur.

ERgene: Python library for screening endogenous reference genes

In gene expression analysis, sample differences and experimental operation differences are common, but sometimes, these differences will cause serious errors to the results or even make the results meaningless. Finding suitable internal reference genes efficiently to eliminate errors is a challenge. Aside from the need for high efficiency, there is no package for screening endogenous genes available in Python. Here, we introduce ERgene, a Python library for screening endogenous reference genes. It has extremely high computational efficiency and simple operation steps. The principle is based on the inverse process of the internal reference method, and the robust matrix block operation makes the selection of internal reference genes faster than any other method.

Selection and validation of optimal endogenous reference genes for analysis of quantitative PCR in four tissues pathologically associated with Kidney-yang deficiency syndrome following influenza A infection.

Kidney-yang deficiency syndrome (KYDS) infected with the influenza virus is a suitable model to imitate a population at high-risk to influenza infection with a high rate of morbidity and mortality. However, the specific molecular mechanisms underlying this disease remain unclear. A stable reference gene is essential as an internal control for molecular biology research of this condition. Reverse-transcription-quantitative PCR (RT-qPCR) is considered an extremely sensitive technique used for absolute and relative quantification of target genes transcript levels. To accurately estimate the relative expression of genes in cells from mice with KYDS in response to infection with influenza A virus subtype H1N1 (A/H1N1) virus using RT-qPCR, it is necessary to identify suitable reference genes. In the present study, analysis of 10 reference genes (Act-β, β2m, GAPDH, Gusβ, Tubα, Grcc10, Eif4h, Rnf187, Nedd8 and Ywhae) was performed across a set of 4 tissue types: Lung; heart; liver; and kidney. KYDS mice were inoculated with A/H1N1 virus or a mock control. For analysis, geNorm, BestKeeper, NormFinder, and Bio-Rad Maestro™ statistical programs were used for the estimation of the stability of the reference genes. The results were authenticated through extended experimental settings using a group of 10 samples, parallel to 3 additional innate immune system-associated genes of the host, TLR3, TLR7 and RIG-I, which were also analyzed using the same algorithms. From the 4 algorithms, taking into account the joint analyses of the ranking order outputs, the 2 genes Ywhae and Nedd8 were identified to be the most stable for mice with KYDS following infection with A/H1N1 virus. In contrast, the least stable genes in all 4 tissues were GAPDH and β2m. These results may affect the choice of reference genes in future studies that use RT-qPCR analysis of target genes in experimental conditions, such as mice with KYDS infected with influenza A virus.

The Expression of MicroRNA-21 in Bone Marrow Fluid is an Indicator of Hematological Disorders and Mortality.

Objective:Bone marrow fluid (BMF) consists of various components that establishes a microenvironment for cell differentiation and remodeling. MicroRNA-21 (miR-21) levels have recently emerged as novel biomarkers for different diseases. However, the conventional RNU6B (U6), used as the reference for intracellular miRNA, may not be appropriate for the normalization of circulating miRNAs. Methods:We measured the levels of U6, spiked-in RNA, and miR-21 in the BMF of 13 healthy controls and 37 patients with hematological disorders to investigate the reliability of either U6 or spike-in RNA as an endogenous reference and also to study the correlation between miR-21, hematological disorders and mortality. Results:Notably, the levels of U6 demonstrated a high variability in BMF of healthy controls and patients. In contrast, the levels of spiked-in RNA displayed a significantly higher stability in both cohorts. Compared with controls, the levels of miR-21 were significantly upregulated in BMF of patients with leukemia but not lymphoma. Also, using 21 as the cut-off value of miR-21, it differentiated the mortality of patients with hematologic disorders. Conclusions:Collectively, using spiked-in RNA as a reference the upregulated miR-21 levels in BMF could be an indicator of the diagnosis of leukemia and a predictor of mortality.