Quantification of mature microRNAs using pincer probes and real-time PCR amplification

PloS One
Tinghua HuangMin Yao

Abstract

The robust and reliable detection of small microRNAs (miRNAs) is important to understand the functional significance of miRNAs. Several methods can be used to quantify miRNAs. Selectively quantifying mature miRNAs among miRNA precursors, pri-miRNAs, and other miRNA-like sequences is challenging because of the short length of miRNAs. In this study, we developed a two-step miRNA quantification system based on pincer probe capture and real-time PCR amplification. The performance of the method was tested using synthetic mature miRNAs and clinical RNA samples. Results showed that the method demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules. The use of pincer probes allowed excellent discrimination of mature miRNAs from their precursors with five Cq (quantification cycle) values difference. The developed method also showed good discrimination of highly homologous family members with cross reaction less than 5%. The pincer probe-based approach is a potential alternative to currently used methods for mature miRNA quantification.

References

May 15, 2002·Current Biology : CB·Mariana Lagos-QuintanaThomas Tuschl
May 16, 2003·Current Biology : CB·Victor AmbrosDavid Jewell
May 24, 2003·Trends in Molecular Medicine·Simone MocellinFrancesco M Marincola
Feb 27, 2004·Nucleic Acids Research·Thomas D SchmittgenLiuqing Yang
Jul 31, 2004·Proceedings of the National Academy of Sciences of the United States of America·George Adrian CalinCarlo M Croce
Apr 27, 2005·Nature Reviews. Molecular Cell Biology·V Narry Kim
Nov 30, 2005·Nucleic Acids Research·Caifu ChenKarl J Guegler
Jan 26, 2006·Nucleic Acids Research·Fuchou TangM Azim Surani
Sep 30, 2006·Analytical Biochemistry·David D DuncanBridget A Lollo
Apr 5, 2007·Nature Protocols·Fuchou TangM Azim Surani
Oct 13, 2007·Plant Methods·Erika Varkonyi-GasicRoger P Hellens
Dec 26, 2007·Methods : a Companion to Methods in Enzymology·Thomas D SchmittgenCaifu Chen
Mar 10, 2009·Molecular Cancer·Amara C SivaMark A Reynolds
Sep 2, 2010·Nature Methods·Monya Baker
Oct 20, 2010·International Journal of Molecular Sciences·Xiaosu WangShenghua Wang
Jun 28, 2011·BMC Biotechnology·Ingrid BalcellsPeter K Busk

❮ Previous
Next ❯

Citations

Sep 29, 2019·BioTechniques·Diego A ForeroGeorge E Barreto
Dec 14, 2018·Laboratory Investigation; a Journal of Technical Methods and Pathology·Vivek Priy DaveDang Duong Bang
Mar 8, 2019·Chemical Science·Lin LanXiangxian Meng

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
electrophoresis
X-ray

Software Mentioned

UNAFold

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.