[Newbie Quick Start] miRNA qPCR detection

The introduction of sequencing technology has prompted the miRNA research field to enter a rapid development stage. However, qPCR is still an important criterion for verifying sequencing data. This article will introduce you to the basics of miRNA analysis using qPCR, and hope to help newcomers get started quickly.

What is a miRNA?

miRNAs are a small class of non-coding RNAs that bind to RNA-induced silencing complexes (RISCs) and act on mRNA to mediate post-transcriptional gene expression. In general, miRNA-RICS complexes are able to inhibit mRNA expression by blocking the translation of the target miRNA or promoting its degradation. Most miRNAs are approximately 18‒22 nucleotides in length and represent only a small fraction of the total RNA in the sample. The amount of miRNA in total RNA is generally considered to be approximately 0.01% (1).

After years of research, the regulation of some mRNAs and their corresponding miRNAs has achieved more data accumulation. Biological processes involved in miRNA include differentiation, development, signal transduction, and host infection responses. In addition, a number of studies have shown that dysregulation of miRNA expression is the cause of diseases such as cancer, or indicators of certain diseases. Circulating miRNA expression is associated with disease and can be used as a source of test samples in serum and plasma, so circulating miRNAs have the potential to become disease-associated biomarkers.

Current miRNA detection technology

To explore the function and post-transcriptional regulation mechanism of miRNA at the cellular level, appropriate detection methods must be used. Currently commonly used methods include quantitative real-time PCR, deep sequencing, and microchips. Each method has its own advantages and disadvantages, which will be explained in detail below.

Quantitative reverse transcription PCR (RT-qPCR)

In an article in the "Analy Review of Analytical Chemistry", " If you choose a method as the gold standard for miRNA detection, then non-RT-qPCR is none other than. (1) "This is because qPCR A number of features meet the key requirements of miRNA detection: this method is accurate and economical, requires no large samples, and has a flexible detection range. When using qPCR to detect RNA, there are several important factors to be aware of, including RNA purity and quality control, cDNA synthesis, primer design, detection, and data homogenization. Data homogenization is more challenging when detecting miRNAs in the blood, and special considerations are needed to achieve effective homogenization and to obtain meaningful data results.

1 First step: cDNA synthesis

The first step in RT-qPCR is to reverse transcribe the RNA extracted from the sample into cDNA. Common problems faced by this step include: (1) the template has only about 22 nucleotides, and (2) the mature miRNA, the precursor miRNA, and the primary transcript coexist.

There are currently two main methods for reverse transcription of miRNA:

• - Reverse transcription using specific primers for different miRNAs

• - Reverse transcription using universal primers for all miRNAs in the sample

Each method has its own advantages and disadvantages. miRNA-specific reverse transcription primers are effective in reducing background noise, while universal primers are suitable for the simultaneous study of multiple different miRNAs. Universal primers usually require the addition of a poly(A) tail and an oligo-dT primer to provide a basis for the simultaneous cDNA synthesis of all miRNAs. QIAGEN's miScript II RT Kit enables easy one-step cDNA synthesis for the detection of all miRNAs in a sample. The kit contains two buffers, miScript HiSpec Buffer and miScript HiFlex Buffer, which specifically convert only mature miRNAs or convert all RNA into cDNA to meet different research needs.

2 Step 2: miRNA-specific primer design

After cDNA synthesis, qPCR can be started. Appropriate primers are required for use in qPCR. The two commonly used miRNA qPCR fluorescent dyes are SYBR Green and dual-labeled hydrolysis probes.

SYBR Green is an embedded dye that binds between the bases of DNA. After binding to dsDNA, the fluorescence intensity is enhanced by about 100-fold, enabling detection of amplification products during PCR. A significant advantage of the SYBR Green method is that there is no need to design specific probes for different miRNAs. However, since the SYBR Green method detects non-specific PCR products and primer dimers, it is necessary to verify the reaction specificity by melting point or melting curve analysis. During the gradual warming of the temperature from 650C to 950C, the fluorescence intensity was recorded, and when the DNA strand began to dissociate, the fluorescence intensity decreased (2). If the melting curve has only one peak, it means that no non-specific amplification product is produced in the reaction.

Unlike the SYBR Green method, dual-labeled hydrolysis probes do not detect non-specific PCR products , but PCR specificity cannot be ignored when using sequence-specific probes - artifacts can result in reduced true PCR product yield. The competition of specific products and artifacts for the reaction components may affect the sensitivity and efficiency of the analysis.

3 The third step: the selection of internal reference RNA and data homogenization

In order to achieve accurate and reproducible miRNA quantification results by real-time PCR, the amount of miRNA to be tested needs to be homogenized using a suitable endogenous reference miRNA. This method is called relative quantification. Normalization avoids inaccurate quantitative results and enables direct comparison between different experiments and different sample test results. The ideal internal reference RNA for homogenization of real-time PCR miRNA detection data should meet the following requirements:

• - All samples involved in the study have stable expression levels

• - size is similar to the measured miRNA

• - the level of expression is similar to the miRNA being tested

• - Expression levels are not regulated under experimental conditions

• - Primers for endogenous reference RNA and miRNA should have similar amplification efficiency (close to 100%)

QIAGEN's miScript PCR Controls meet the above requirements for accurate and reliable relative quantification of miRNAs from multiple species such as humans, rats, mice and dogs by the ΔΔCT method.

QIAGEN has a comprehensive solution for the quantitative and functional research of non-coding RNAs such as miRNAs. It is suitable for the comprehensive study of miRNAs in single-cell, circulating body fluids, FFPE, exosomes and other difficult samples. The detection system is simple and completely Experimental verification ensures that the results are accurately obtained.

Second generation sequencing/RNA-seq

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