Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification

Abstract

MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of seven logs and a sensitivity sufficient to detect down to ten target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (r2 = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

“…Moreover, multiple fluorescence signals can be used to detect the expression of several genes simultaneously using multiple channels . Quantitative PCR using TaqMan can be used to evaluate the normal control group and cancer groups in the same qPCR tube using the same cDNA template . Additionally, TaqMan probes can increase the specificity and sensitivity of qPCR and reduce system error in the experiment, providing a good basis for the evaluation of clinical application value.…”

CRISPR/Cas13a‐Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification‐Free miRNA Diagnostics

“…Today, there are a few reports tackling these challenges, for example, by improving current detection methods, using electrochemical assays or developing pH‐based POC tests . However, these approaches still need error‐prone amplification steps prior to the miRNA detection, have rather tedious preparation steps, or are limited by the design of suitable primers . To address these issues, we combine microfluidics with an electrochemical signal readout to develop a sensitive (i.e., target amplification‐free) and selective diagnostic test, while enabling a miniaturization for POC testing .…”

“…[9] However, these approaches still need error-prone amplification steps prior to the miRNA detection, have rather tedious preparation steps, or are limited by the design of suitable primers. [10,11] To address these issues, we combine microfluidics with an electrochemical signal readout to develop a sensitive (i.e., target amplification-free) and selective diagnostic test, while enabling a miniaturization for POC testing. [12,13] We further apply the newly discovered CRISPR/Cas13a technology, which is able of targeting almost any RNA, to our developed electrochemical biosensor, creating a novel and powerful tool for miRNA diagnostics (Figure 1a).…”

Evaluation of miRNA Detection Methods for the Analytical Characteristic Necessary for Clinical Utilization

“…Unlike miR-142-5p and miR-93-5p, this miR-23a-3p is a member of a larger family that also includes miR-23b and miR-23c (www.mirbase.org). Measurement of a miRNA that has another sibling is always more complicated in terms of specificity [25]. This could cause greater variation in measurements by different technologies and, as a consequence, lower correlation results [26].…”