miRNA assay
ALMIR – miRNA Assay Kits
A modern technological solution for basic research and development of new clinical diagnostic methods!
miRNAs are short ribonucleic acid molecules (20–22 nucleotides) that regulate genome expression on post-transcription level. Alteration of miRNA regulatory function is strongly associated with cancer development. Therefore, miRNAs are considered as promising markers for cancer diagnostics as well as targets for anticancer therapy.
The individual gene expression blocking is miRNA general mechanism. In 2006 American scientists Andrew Fire and Craig Mello received the Nobel Prize in Physiology and Medicine for describing this phenomenon. Over the next years, our knowledge of the gene expression regulation system with the help of miRNA molecules has significantly extended. It is suggested that the complex system of these regulatory molecules interaction is the general mechanism for the dynamic regulation of the protein-secreting cell apparatus.
Neoplastic transformation occurs due to a composition change and, thus, due to the miRNA apparatus regulatory activity distortion. Moreover, this causal intercommunication can have different cause-effect relations.
It was noticed that the genome fragile sites – the sites of frequent aberrations and mutations – often encode entire miRNA molecules families. Mutations in these sites distort the composition of cellular miRNAs and lead to the development of various oncological diseases, including lung, colon and stomach cancer. This points to a possible etiological role of miRNA in cancer development. In addition, miRNAs in a normal cell regulate metabolism, proliferative activity, differentiation, and scheduled cell death (apoptosis). Disruption of these processes during neoplastic transformation can cause adaptive (secondary) changes in the intracellular miRNAs profile. The cancerogenic nature of some miRNAs has been confirmed by many studies. For example, miR-21, a miRNA molecule that inhibits the production of a number of tumor suppressor proteins by a cell, is involved in the development of many tumors. In modern molecular oncology, the concept of “onco-miR” has appeared—a miRNA with a distinctive cancerogenic effect, for example miR-155, a cluster of related molecules miR-106b / miR-17–92.
Neoplastic transformation of cells is accompanied by specific changes in the profile (composition) of intracellular miRNAs, which determines its potential diagnostic value. miRNAs isolation from biopsy specimen (fresh, fixed, stained) and analysis of the marker miRNA molecule profile is a promising diagnostic test. Moreover, the features of the miRNA profile can be associated with certain clinically significant characteristics of the tumor (for example, sensitivity to certain therapy), which can be used for personalization of treatment.
A modern technological solution for basic research and development of new clinical diagnostic methods
The developed technology is based on a method proposed by a group of researchers led by Mikael Kubista in 2017. We’ve modified the original method to suite the routine work at your convenience. Original design of two-claws primer for reverse transcription, optimized enzyme mixes and fluorescent probe – based detection of amplification determine the high accuracy and sensitive of the analytic system.
Improved specificity
In comparison to traditional methods, the technology of two-claws RT-qPCR provides with high specificity of miRNA binding and reverse transcription. In addition to this property, ALLMIR kits employ fluorescent-labelled probe for miRNA-specific detection of amplification. This defined exceptional specificity of analysis.
Absolute quantification
ALLMIR scramble systems are developed to be used in parallel with assayed miRNA and to allow quantitative analysis. Scramble-RNAs of known concentrations are being added in RNA samples before reverse transcription. Results of qPCR analysis of scramble-RNA mixed in real sample are used as calibrators for absolute quantification of miRNA of interest.
Controlled sensitivity
Any analytic method has certain limit of detection. When miRNA is assayed by any of RT-qPCR approach, the interaction of PCR primers with partially complement RT primer might result into false positive signal detected at the late cycles of PCR. ALLMIR kits provides with option to test if the sensitivity of system is sufficiently high to estimated concentration of low-copied miRNA in your sample. Each kit contains eight calibrators (set of dilutions of synthetic analog of the miRNA), that can be used to check operability of the system. Confident results of biological samples analysis must be obtained within diapason of linear dependency of miRNA concentration and amplification rate (threshold cycle, Ct).
X‑direction: concentration of synthetic miR-371–3p molecules in the RT reaction mixture (20µL), 10^
Method
RT-qPCR is the common approach for miRNA analysis in laboratory practice due to its low-cost and reliability. The kit implements the original technology of two-claws reverse transcription followed by quantitative PCR with two miRNA-specific primers, that provides exceptional accuracy of analysis.
Principles of analysis
Despite its widespread use, the practical use of the RT-PCR method is a sophisticated task and following aspect must be addressed
1. The results of the quantitative analysis of the miRNA significantly depend on the quality of the RNA isolated from biological samples.
2. Due to short length the mature miRNA molecule not only undergoes reverse transcription but also elongation. These modifications are necessary to enable the next step of quantitative PCR.
3. The presence of very similar or immature form of miRNA can interfere with RT-qPCR results.
Assay duration
Material
Two phases of miRNA assay
Reverse transcription (RT)
Synthesis of cDNA, or reverse transcription (RT), is carried out using a miRNA-specific primer with two sites of complementary interaction with a miRNA molecule. The synthesis of complementary chain is started from 3’-craw of RT primer while 5’-craw is displaced. Resulted molecule of cDNA has a length 60–70 nucleotides and is flanked by two miRNA-specific sites for binding by PCR-primers.
Ready to Use Reagents for Reverse Transcription
RT enzyme
RT bufer
Contains a mixture of Tris-HCl (pH 8.3), KCl, MgCl2, dNTPs, dithiothritol. The buffer contains a unique miRNA-specific RT-primer.
Polymerase chain reaction (PCR)
The polymerase chain reaction is performed using two miRNA-specific primers. The amplification efficiency is assessed in real-time (real-time quantitative PCR) using miRNA-specific probes with a dye label (FAM) and appropriate quencher.
Ready to Use Reagents for PCR
PCR enzyme
PCR bufer
