Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA

Yang, Lihong; Wang, Silu; Tang, Lili; Liu, Biao; Ye, Wen‑Le; Wang, Lingling; Wang, Zhang-yang; Zhou, Mengtao; Chen, Bicheng

doi:10.1371/journal.pone.0115293

Cited by 69 publications

(41 citation statements)

References 18 publications

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“…Specificity and reliability in the present study were guaranteed with the use of a stem-loop RT primer and TaqMan probe. Due to the advantage of the high specificity of the stem-loop RT primer, it was used to perform the reverse transcription of miRNA (34,35). The TaqMan probe was utilized in the PCR process since the accuracy of TaqMan RT-qPCR is significantly higher than when using SYBR-Green in PCR analysis (36,37).…”

Section: Discussionmentioning

confidence: 99%

Clinical evaluation of 4 types of microRNA in serum as biomarkers of esophageal squamous cell carcinoma

et al. 2018

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To the best of our knowledge, there is currently no specific biomarker for esophageal cancer used in clinical practice. However, studies consider that microRNAs (miRNAs/miRs) could have useful implications in clinical practice. The present study aimed to investigate the feasibility of using serum microRNAs as biomarkers for esophageal squamous cell carcinoma (ESCC). Using reverse transcription-quantitative polymerase chain reaction, the expression levels of serum miR-21, miR-25, miR-145 and miR-203 were detected in 31 untreated patients with ESCC (EC-UT), 35 inactive period patients with ESCC following treatment (EC-T), 33 patients with esophageal benign disease (benign) and 32 healthy donors (healthy). Furthermore, the ability of these microRNAs to function as biomarkers of ESCC alone and in combination were investigated. The expression levels of serum miR-21, miR-25 and miR-145 in EC-UT were significantly higher than in the other groups (P<0.001). High sensitivity and specificity were shown when miRNAs were used as biomarkers for ESCC, particularly miR-21 and the combination of miR-21 with miR-145. Comparing EC-UT with healthy, benign and EC-T groups, and a combined group (3 groups set as 1 negative control), the sensitivity and specificity of miR-21 were 71.0 and 96.9, 74.2 and 87.9, 77.4 and 82.9, and 74.2 and 88.0%, respectively. The combined sensitivity and specificity of miR-21 and miR-145 were 71.0 and 96.9, 90.9 and 72.7, 97.1 and 82.9, and 80.6 and 80.0%, respectively. In conclusion, 3 types of miRNA (miR-21, miR-25 and miR-145) in serum could serve as potential biomarkers for ESCC. Furthermore, the expression level of miR-145 in serum was upregulated, compared with the downregulation reported in previous studies in ESCC tissues and cells.

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Section: Discussionmentioning

confidence: 99%

Clinical evaluation of 4 types of microRNA in serum as biomarkers of esophageal squamous cell carcinoma

et al. 2018

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“…In this sense, the stem-loop RT-qPCR technique has proven to be highly specific to detect and suitable to quantify specific small RNAs [31,32,33,36,53,57]. Moreover, this technique is fast and easy to perform and has a lower cost compared with Northern blotting.…”

Section: Discussionmentioning

confidence: 99%

“…lamblia , fluorescence in situ hybridization (FISH) was used to find the cellular location of the snoRNA GlsR17 and its product, miR2 [6]; however, these techniques require time, and they can be laborious, expensive, and have low sensitivity [27]. Stem-loop quantitative reverse transcription PCR (RT-qPCR) is an efficient and novel strategy that has been used in recent years to detect and amplify mature miRNAs [31,32,33,34]. The stem-loop is designed such that it is shaped as a hairpin and possesses a 3′ overhang complementary to the miRNA.…”

Section: Introductionmentioning

confidence: 99%

Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia

Marcial-Quino

Gómez-Manzo

Fierro

et al. 2016

Genes

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Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect—in a rapid, sensitive, specific, and reproducible way—the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia.

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“…The method for qRT-PCR for Rpl13a snoRNAs used target-specific stem loop RT primers, a targetspecific forward PCR primer, and a universal reverse PCR primer (46). This method can detect as few as 10 3 copies of target RNAs, is able to distinguish between small RNA species that differ by only 1 nucleotide, is not affected by genomic contamination, distinguishes mature snoRNAs and miRNAs from their precursor small RNA species, and has been validated against RNA-Seq for snoRNAs (10,47,48). mRNA quantification was calculated using Rplp0 as an endogenous control.…”

Section: Qrt-pcrmentioning

confidence: 99%

Long-range function of secreted small nucleolar RNAs that direct 2′-O-methylation

Rimer

Lee

Holley

et al. 2018

Journal of Biological Chemistry

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Small nucleolar RNAs (snoRNAs) are noncoding RNAs that guide chemical modifications of structural RNAs. Whereas snoRNAs primarily localize in the nucleolus, where their canonical function is to target nascent ribosomal RNAs for 2'--methylation, recent studies provide evidence that snoRNAs traffic out of the nucleus. Furthermore, RNA-Seq data indicate that extracellular vesicles released from cells contain snoRNAs. However, it is not known whether snoRNA secretion is regulated or whether secreted snoRNAs are functional. Here, we show that inflammation stimulates secretion of snoRNAs U32a (SNORD32a), U33 (SNORD33), U34 (SNORD34), and U35a (SNORD35a) from cultured macrophages, in mice, and in human subjects. Secreted snoRNAs co-fractionate with extracellular vesicles and are taken up by recipient cells. In a murine parabiosis model, we demonstrate that snoRNAs travel through the circulation to function in distant tissues. These findings support a previously unappreciated link between inflammation and snoRNA secretion in mice and humans and uncover a potential role for secreted snoRNAs in cell-cell communication.

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Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA

Cited by 69 publications

References 18 publications

Clinical evaluation of 4 types of microRNA in serum as biomarkers of esophageal squamous cell carcinoma

Clinical evaluation of 4 types of microRNA in serum as biomarkers of esophageal squamous cell carcinoma

Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia

Long-range function of secreted small nucleolar RNAs that direct 2′-O-methylation

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