In gene expression research, microRNAs (miRNAs) pose a challenge to conventional fluorescent quantitative detection due to their short chain nature (approximately 22nt in length). How to accurately quantify these small molecules is a key issue faced by researchers. At present, the mainstream technological paths are divided into the stem loop method and the tail addition method, each with its own advantages. The choice of which path depends on the specific needs of the experiment.

From a technical perspective, both are aimed at solving the challenge of designing miRNA reverse transcription primers.Stem-loopUsing a primer with a special stem loop structure for reverse transcription, this primer can specifically bind to the 3 'end of miRNA and increase the length of the reverse transcription product through its stem loop structure, providing convenience for subsequent qPCR amplification. Its core advantage lies inHigh specificityIt is considered the quantitative "gold standard" as it can effectively distinguish highly homologous miRNA family members and even recognize differences in individual bases.

ButTail addition methodThen another strategy is adopted: it first adds a string of adenine tail (PolyA tail) to the 3 'end of miRNA through PolyA polymerase, and then uses universal Oligo (dT) primers for reverse transcription. The characteristics of this method areEasy to operate and high throughputWith just one tail reaction, all miRNAs can be reverse transcribed, making it suitable for large-scale screening or sample size pre experiments.

In practical applications, the key to decision-making lies in balanceSpecificity and fluxIf the research objective is to validate specific and known miRNAs, and there are strict requirements for distinguishing subtle differences between family members (such as disease marker screening), the stem loop method is preferredgoodSpecificity is a more reliable choice. For example, in relevant miRNA fluorescence quantitative detection services, in order to ensure absolute accuracy of individual gene expression data in clinical research or basic scientific research, technology providers often prioritize recommending the stem loop method.

On the contrary, if the research is in the early stage of discovery and requires simultaneous detection of the expression profiles of hundreds of miRNAs in a large number of samples, or if the total amount of RNA in the samples is extremely limited, then the tail addition method, with its advantages of short operation process and low required RNA amount, can provide a global overview of expression data more quickly.

In summary, the stem loop method and tail addition method are not substitutes, but complementary technical means. A wise choice stems from a clear understanding of the experimental objective: whether to pursue the accuracy of individual data or explore the overall expression of the group. Only by combining specific needs can the correct path to reliable results be selected.

MiRNA fluorescence quantitative detection service

Product Introduction

Real Time PCR， Real time fluorescence quantitative PCR, also known as quantitative PCR or qPCR,It is a method of using fluorescent chemicals to measure each amplification in DNA amplification reactionspolymerase chain reactionThe method of determining the total amount of products after the PCR cycle. A method for quantitative analysis of specific DNA sequences in a test sample using internal or external reference methods

Due to its advantages of simple operation, fast and convenient, high sensitivity, good repeatability, and low contamination rate, quantitative PCR is widely used in various fields such as medical testing, drug efficacy assessment, gene expression research, transgenic research, gene detection, pathogen detection, animal and plant detection, food detection, etc

Experimental Principle

The so-called real-time fluorescence quantitative PCR technology refers to the method of adding fluorescent groups to the PCR reaction system, using fluorescence signal accumulation to monitor the entire PCR process in real time, and finally quantitatively analyzing unknown templates through standard curves

Overview of real-time fluorescence quantitative PCR technology types: DNA binding dye method, probe based chemical method, quenching dye primer method

Experimental application

DNA and RNA quantification; Gene expression validation; Allele identification; SNP typing; Pathogen and virus detection; Multiplex PCR

experimental results