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

2019 ◽  
Vol 20 (18) ◽  
pp. 4353 ◽  
Author(s):  
Simone Donati ◽  
Simone Ciuffi ◽  
Maria L. Brandi
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Biological Fluids ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Transcriptional Level ◽  
Circulating Mirnas ◽  
Endogenous Reference ◽  
Mirnas Expression ◽  
Endogenous Reference Genes

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.

scholarly journals Identification of Endogenous Controls for Analyzing Serum Exosomal miRNA in Patients with Hepatitis B or Hepatocellular Carcinoma

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Yi Li ◽  
Liqun Zhang ◽  
Fei Liu ◽  
Guiming Xiang ◽  
Dongneng Jiang ◽  
...  
Keyword(s):  
Reference Genes ◽  
Early Stage ◽  
Quantitative Polymerase Chain Reaction ◽  
Control Gene ◽  
Exosomal Mirnas ◽  
Endogenous Reference ◽  
Polymerase Chain ◽  
Noninvasive Biomarkers ◽  
Exosomal Mirna ◽  
Endogenous Reference Genes

Serum exosomal microRNAs (miRNAs) have received considerable attention as potential biomarkers for diagnosing cancer. The canonical technique for measuring miRNA transcript levels is reverse transcription quantitative polymerase chain reaction (RT-qPCR). One prerequisite for validating RT-qPCR data is proper normalization with respect to stably expressed endogenous reference genes. However, genes that meet all of the criteria of a control gene for exosomal miRNAs have not yet been identified. To find out the control gene for exosomal miRNAs, we evaluated the expression stability of 11 well-known reference genes in circulating exosomes. In this study, we found that the combination ofmiR-221,miR-191,let-7a,miR-181a, andmiR-26acan be an optimal gene reference set for normalizing the expression of liver-specific miRNAs. This combination enhanced the robustness of the relative quantification analyses. These findings highlight the importance of validating reference genes before quantifying target miRNAs. Furthermore, our findings will improve studies that monitor hepatitis progression and will aid in the discovery of noninvasive biomarkers to diagnose early stage HCC.

scholarly journals Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle,Lethrus apterus(Coleoptera: Geotrupidae)

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4047 ◽  
Author(s):  
Nikoletta A. Nagy ◽  
Zoltán Németh ◽  
Edit Juhász ◽  
Szilárd Póliska ◽  
Rita Rácz ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Real Time ◽  
Reference Genes ◽  
Hormonal Regulation ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Body Region ◽  
Body Regions ◽  
Polymerase Chain

Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples ofLethrus apterus, an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. Additionally, L7A and RP18 appear to be the best reference genes for normalisation in all samples irrespective of body region. These reference genes can be used to study the hormonal regulation of reproduction and parental care inLethrus apterusin the future.

scholarly journals Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae)

2017 ◽  
Author(s):  
Nikoletta A Nagy ◽  
Zoltán Németh ◽  
Edit Juhász ◽  
Szilárd Póliska ◽  
Rita Rácz ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Real Time ◽  
Reference Genes ◽  
Hormonal Regulation ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Body Regions ◽  
Qpcr Analysis ◽  
Polymerase Chain

Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors in question by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of Lethrus apterus, an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. These reference genes can be used to study the hormonal regulation of reproduction and parental care in Lethrus apterus in the future.

scholarly journals Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae)

2017 ◽  
Author(s):  
Nikoletta A Nagy ◽  
Zoltán Németh ◽  
Edit Juhász ◽  
Szilárd Póliska ◽  
Rita Rácz ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Real Time ◽  
Reference Genes ◽  
Hormonal Regulation ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Body Regions ◽  
Qpcr Analysis ◽  
Polymerase Chain

Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors in question by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of Lethrus apterus, an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. These reference genes can be used to study the hormonal regulation of reproduction and parental care in Lethrus apterus in the future.

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