Reference microRNAs Research Articles

In-Depth Characterization of the MicroRNA Transcriptome in Normal Thyroid and Papillary Thyroid Carcinoma

A single microRNA gene may give rise to several mature products that differ in length, called isomiRs. IsomiRs are known to be tissue specific and functionally relevant. The microRNA sequence heterogeneity of the thyroid gland has yet to be determined. The objective of the study was to provide a comprehensive view of the microRNA transcriptome in normal thyroid and papillary thyroid carcinoma (PTC). We used next-generation deep sequencing to analyze microRNA length heterogeneity and expression profiles of PTC tumors (n = 14), unaffected tissue adjacent to tumors (n = 14), and control, noncancerous thyroid tissue (n = 14). The results were validated with a microarray on an additional set of 9 PTC tumor/normal tissue pairs. Eighty-nine microRNAs were significantly deregulated in PTC compared with normal thyroid tissue (false discovery rate < 0.05, fold change 0.13-20.7). Top deregulated miRNAs included miR-146b-5p, miR-221-3p, miR-7-3p, miR-551b-3p, miR-486-3p, and miR-144-3p, confirming previous microarray profiling. The expression of miRNAs did not depend on the BRAF mutation status. Interestingly, 85% of the most abundant microRNAs consisted of isoforms that differed from the standard reference sequence deposited in miRBase. Moreover, the reference microRNAs were completely absent in 42.4% and 35.9% of the microRNAs expressed in normal thyroid and PTC tumors, respectively. Numerous isomiRs had altered seed sequences, which led to a different set of target genes. For highly deregulated miR-146b-5p, we detected 6 isoforms (tumor/normal fold change 14.4-28.7, false discovery rate < 0.002) that varied at their 5' ends with a 1-nt difference that created 2 alternative seeds. The target genes for those 2 seeds overlapped in only 13.1% of genes. Almost all microRNAs exhibit isoforms of variable length and potentially distinct function in thyroid tumorigenesis.

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation

AbstractAssessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at −180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at −20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

The Repertoire and Features of Human Platelet microRNAs

Playing a central role in the maintenance of hemostasis as well as in thrombotic disorders, platelets contain a relatively diverse messenger RNA (mRNA) transcriptome as well as functional mRNA-regulatory microRNAs, suggesting that platelet mRNAs may be regulated by microRNAs. Here, we elucidated the complete repertoire and features of human platelet microRNAs by high-throughput sequencing. More than 492 different mature microRNAs were detected in human platelets, whereas the list of known human microRNAs was expanded further by the discovery of 40 novel microRNA sequences. As in nucleated cells, platelet microRNAs bear signs of post-transcriptional modifications, mainly terminal adenylation and uridylation. In vitro enzymatic assays demonstrated the ability of human platelets to uridylate microRNAs, which correlated with the presence of the uridyltransferase enzyme TUT4. We also detected numerous microRNA isoforms (isomiRs) resulting from imprecise Drosha and/or Dicer processing, in some cases more frequently than the reference microRNA sequence, including 5′ shifted isomiRs with redirected mRNA targeting abilities. This study unveils the existence of a relatively diverse and complex microRNA repertoire in human platelets, and represents a mandatory step towards elucidating the intraplatelet and extraplatelet role, function and importance of platelet microRNAs.

MicroRNAs are suitable for assessment as biomarkers from formalin-fixed paraffin-embedded tissue, and miR-24 represents an appropriate reference microRNA for diffuse large B-cell lymphoma studies

Tissue biopsy specimens in the form of formalin-fixed paraffin-embedded tissue (FFPET) represent a valuable resource for biomarker identification and validation. However, to date, they remain an underused asset due to...

Determination of Reference microRNAs for Relative Quantification in Porcine Tissues

Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r2 = 0.99). The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs (miR-93, miR-25, miR-106a, miR-17-5p and miR-26a) in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies (let-7a, miR-17-5p and miR-25 in skeletal muscle; miR-17-5p, miR-93 and miR-26a in kidney, miR-26a, miR-103 and let-7a in liver, let-7a, miR-25 and miR-106a in ovary and miR-103, let-7a and miR-93 in uterus). Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken.

Identification of prostate cancer-expressed microRNAs associated with clinical recurrence (cR) and prostate cancer-specific survival (PCSS) following radical prostatectomy (RP).

21 Background: We previously identified messenger RNAs (mRNAs) whose expression can distinguish aggressive from indolent prostate cancer. Representing multiple key genomic pathways, these mRNAs are significantly associated with cR and PCSS after RP, providing prognostic information beyond PSA, cT stage and Gleason Score. We evaluated microRNA expression in the same specimens for association with cR and PCSS. Methods: All cT1/cT2 prostate cancer pts treated with RP at Cleveland Clinic from 1987-2004 were identified (n~2,600), of which 127 pts w/ cR and 374 pts w/o cR after RP were randomly selected using cohort sampling. RNA was purified from 2 macrodissected FPE tumor specimens per pt. Expression of 76 test and 5 reference microRNAs was quantified by RT-PCR. Cox regression and control of the false discovery rate (FDR) was used to assess reference-normalized microRNA and mRNA expression for association with cR and PCSS. Results: 106 pts with cR and 310 without cR had sufficient RNA and successful microRNA assays. Analysis of primary Gleason pattern tumor tissue for each pt identified 21 microRNAs associated with cR and 13 microRNAs associated with PCSS, with FDR at 10%; 8 microRNAs were associated with both endpoints. Similar analysis of highest Gleason pattern tumor tissue for each pt identified 22 microRNAs associated with cR (17 overlapping with those for the primary Gleason pattern) and 7 microRNAs associated with PCSS, with FDR at 10%; 4 were associated with both endpoints. miR-1, miR-21, miR-93, and miR-106b were associated with both cR and PCSS in primary and highest Gleason pattern specimens. The 76 microRNAs in this study tended to have weaker association with cR and PCSS than the 732 mRNAs. In multivariate analyses, mRNAs and microRNAs provided prognostic information beyond PSA, cT stage, and biopsy Gleason score. MicroRNAs co-express more frequently with each other than with mRNAs, which may indicate distinct biological regulation. Conclusions: Expression of some microRNAs assayed in FPE prostate tumor tissue was associated with cR and PCSS after RP in this study, and may retain prognostic value in the face of tumor heterogeneity.

Identification of reference microRNAs and suitability of archived hemopoietic samples for robust microRNA expression profiling

In many cancers, including neuroblastoma, microRNA (miRNA) expression profiling of peripheral blood (PB) and bone marrow (BM) may increase understanding of the metastatic process and lead to the identification of clinically informative biomarkers. The quality of miRNAs in PB and BM samples archived in PAXgene™ blood RNA tubes from large-scale clinical studies and the identity of reference miRNAs for standard reporting of data are to date unknown. In this study, we evaluated the reliability of expression profiling of 377 miRNAs using quantitative polymerase chain reaction (qPCR) in PB and BM samples (n=90) stored at −80°C for up to 5years in PAXgene™ blood RNA tubes. There was no correlation with storage time and variation of expression for any single miRNA (r<0.50). The profile of miRNAs isolated as small RNAs or co-isolated with small/large RNAs was highly correlated (r=0.96). The mean expression of all miRNAs and the geNorm program identified miR-26a, miR-28-5p, and miR-24 as the most stable reference miRNAs. This study describes detailed methodologies for reliable miRNA isolation and profiling of PB and BM, including reference miRNAs for qPCR normalization, and demonstrates the suitability of clinical samples archived at −80°C into PAXgene™ blood RNA tubes for miRNA expression studies.

Identification of miR-23a as a novel microRNA normalizer for relative quantification in human uterine cervical tissues

Quantitative real-time RT-PCR (RT-qPCR) is being widely used in microRNA expression research. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in microRNA RT-qPCR studies. The aim of this study was to identify the most stable reference gene(s) for quantification of microRNA expression analysis in uterine cervical tissues. A microarray was performed on 6 pairs of uterine cervical tissues to identify the candidate reference genes. The stability of candidate reference genes was assessed by RT-qPCR in 23 pairs of uterine cervical tissues. The identified most stable reference genes were further validated in other cohort of 108 clinical uterine cervical samples: (HR-HPV- normal, n=21; HR-HPV+ normal, n=19; cervical intraepithelial neoplasia [CIN], n=47; cancer, n=21), and the effects of normalizers on the relative quantity of target miR-424 were assessed. In the array experiment, miR-26a, miR-23a, miR-200c, let-7a, and miR-1979 were identified as candidate reference genes for subsequent validation. MiR-23a was identified as the most reliable reference gene followed by miR-191. The use of miR-23a and miR-191 to normalize expression data enabled detection of a significant dereg-ulation of miR-424 between normal, CIN and cancer tissue. Our results suggested that miR-23a and miR-191 are the optimal reference microRNAs that can be used for normalization in profiling studies of cervical tissues; miR-23a is a novel microRNA normalizer.

Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes

MicroRNAs (miRNAs) constitute a group of small non-coding RNA molecules generally 18–22 base-pairs in length. miRNAs are considered to be negative regulators of gene expression at the level of post-transcription and are important in normal physiological development, tissue homeostasis and disease. The cellular origin of individual microRNAs is of utmost importance for understanding their roles in molecular and biological processes, in multi-cellular and complex structured tissues. For the localization of miRNAs in clinical and experimental formalin-fixed and paraffin-embedded samples we have developed a simple and robust one-day in situ hybridization protocol based on the use of double digoxigenin (DIG)-labeled LNA–DNA chimeric probes. We show that the protocol enables analyses of specificity, and demonstrate the detection of miR-1, miR-21, miR-124, miR-126, miR-145, and miR-205 in human and murine paraffin material. The well established localization of these microRNAs makes them ideal for use as reference microRNAs when optimizing the microRNA in situ hybridization assay for a particular tissue and miRNA.

Quality Assessment and Data Analysis for microRNA Expression Arrays

MicroRNAs are small (∼22 nt) RNAs that regulate gene expression and play important roles in both normal and disease physiology. The use of microarrays for global characterization of microRNA expression is becoming increasingly popular and has the potential to be a widely used and valuable research tool. However, microarray profiling of microRNA expression raises a number of data analytic challenges that must be addressed in order to obtain reliable results. We introduce here a universal reference microRNA reagent set as well as a series of nonhuman spiked-in synthetic microRNA controls, and demonstrate their use for quality control and between-array normalization of microRNA expression data. We also introduce diagnostic plots designed to assess and compare various normalization methods. We anticipate that the reagents and analytic approach presented here will be useful for improving the reliability of microRNA microarray experiments.