Kits for basic development

miRNA assay

miRNAs are short ribonucleic acid molecules (containing 20–22 nucleotides). Its chemical structure is similar to well-studied messenger, transfer and ribosomal RNA molecules.   To date, it has been estimated that there are about 3,000 types of miRNA molecules in human cells and that they regulate most protein-coding genes.

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.

miRNA Assay Kits

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. Different RT primer structure, changed technology for assessing the real-time amplification efficiency and optimized conditions for the enzymes have made the work of the system more reliable and sensitive – all the changes allow to make a reliable quantitative evaluation of the selected microRNA molecule in a wide concentration range.

Assay sensitivity

RT primer structure modification made it possible to use probes labeled with a fluorochrome (carboxyfluorescein, FAM) and generating a fluorescent signal after complementary interaction with the amplicon. This technology for assessing the amplification efficiency determines high sensitivity and additional assay specificity.

Assay reliability

Each kit contains eight calibrators (synthetic analog solutions of the detected miRNA molecule of various concentrations), that provide operability and confidence in the results obtained. Calibrators help every user to be able to check system health and RT-PCR methodological results reproducibility.

Assay without normalization

The possibility to quantitatively evaluate miRNA in RNA samples isolated from biological material appears after a calibration curve deriving. The calibration curve directly correlates amplification efficiency (threshold cycle, Ct) with analyte concentration (miRNA).

The concentration evaluation of miRNA under study might be carried out directly, without using the “normalization” procedure as to conditional «normalizers» (U6, U18, RNU44, RNU48, etc.), if the standardized RNA extraction protocol is used and Ct values in biological sample analysis fit into a linear dependency zone.

Концентрация молекул микроРНК от порогового цикла
Y‑direction: hreshold cycle, Ct
X‑direction: кoncentration of synthetic miR-371–3p molecules in the reaction mixture, 10^

Assay methods

RT-PCR is the main miRNA assay method in laboratory practice due to its reliability and economic efficiency.

The kit implements the original technology of reverse transcription two-way priming and PCR with two miRNA-specific primers (Androvic et al., 2017), that provides exceptional analysis accuracy.

Reliable results and higher assay sensitivity (compared to other technologies, Korobkina et al., 2018) are provided by RT primer structure optimization and amplification detection with a probe. This allows to work with low concentration RNA samples.

Kit components

Inland manufactured optimized Enzyme Blends for RT and PCR.

Balanced mixtures of oligonucleotides for priming reactions and detecting the real-time amplification process.

A set of 8 calibrators—dilutions of synthetic “mimic” for making a calibration curve and miRNA assay in biological material.

Assay duration

Assay duration: 2.5 – 3 hours, including RT (45 «) and PCR (60»).

Material

Material requirements: RNA must be isolated (from cells, tissues, biological fluids) using methods that exclude the short molecules «loss».
Extracellular miRNAs can be present in many biological fluids, including plasma. Therefore, the assessment of qualitative or quantitative changes in the circulating miRNA composition is one of the promising directions in the fluid biopsy development.

Two phases of miRNA assay

Microarray technology, sequencing, including genome-wide RNA sequencing, and reverse transcription followed by polymerase chain reaction (RT-PCR) – the same methods that are traditionally used for the messenger RNA assay – are fundamentally applicable for the miRNA assay. Currently, RT-PCR is the most commonly used technique due to its reliability and relatively low cost.

Despite its widespread use, the practical use of the RT-PCR method is a sophisticated task. 

  1. The miRNA assay results significantly depend on the quality of the RNA isolated from biological samples.
  2. The RT-PCR results can be distorted by the presence of molecules immature forms.
  3.  
  4. The length of the mature miRNA molecule is comparable in size to the length of the primers, the pair of which must have “landing sites” along the detected molecule to initiate the polymerase chain reaction.
Therefore, before performing analytical PCR, it is necessary to «elongate» the miRNA molecule. This stage of the DNA molecule synthesis, that should be at least 50–60 bases in length and part of which is complementary to the detected miRNA, is a critical aspect of the technique for miRNA assay using RT-PCR.

Reverse transcription (RT)

Synthesis of DNA, that is partially complementary and detectable to a miRNA molecule, or reverse transcription (RT), is carried out using a miRNA-specific primer with two sites of complementary interaction with a miRNA molecule. As a result of the reaction, a molecule with a length of 65–75 nucleotides is synthesized.
miRNA reverse transcription method using a primer that forms the so-called stem-loop primer was proposed in 2005. This approach made it possible, as a result of the reverse transcription reaction, to obtain cDNA with a length of 50–60 bases, which provides the possibility of using such molecule as a template for subsequent PCR. Over the next years, this method has been optimized by many scientists. And now it is widely used and provides reliable results. The molecular specificity of the reverse transcription reaction is an important feature of this approach. An appropriate RT primer is synthesized and used for each type of detected miRNA.
Синтез молекулы комплементарной или частично комплементарной ДНК
Later, several more methods for the cDNA synthesis were proposed. Their main feature was the nonspecific «elongation» of all miRNA molecules present in the sample. The subsequent reverse transcription reaction is initiated by a primer complementary to the added site. Such reaction is nonspecific and, in theory, makes it possible to obtain a pool of different cDNAs corresponding to the set of miRNAs in the study sample. On the one hand, this approach allows economical use of biological material for the analysis of a large number of different miRNA molecules. On the other hand, the sensitivity of this method is lower because the reverse transcription reaction is initiated using a universal primer equally probable for all miRNA molecules, which can complicate the detection of molecules with low copy number.
Обратная транскрипция кДНК | Альгимед Техно

Ready to Use Reagents for Reverse Transcription

RT enzyme

Genetically modified reverse transcriptase (revertase) from murine leukemia virus (M‑MuLV). As a result of the modification, the enzyme retained its RNA-dependent polymerase activity, but lost the wild-type RNase activity. The enzyme is able to synthesize the cDNA strand; it exhibits optimal activity at 42 ° C.

RT bufer

Contains a mixture of Tris-HCl (pH 8.3), KCl, MgCl2, dNTP mixture, dithiothritol. The buffer contains a unique miRNA-specific RT-primer for obtaining a cDNA molecule partially complementary to the detected miRNA and having a length of 65–75 nucleotides.

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 probes with a dye label (FAM).
An original «elongation» and reverse transcription method was recently proposed by a group of Czech scientists. This method involves the use of a relatively long stem-loop primer for RT, and its two ends complementarily bind the ends of the detected miRNA, being located «towards» each other. The RT reaction is initiated by one of the primer ends (3′-). In the process of transcription, the second end (5′-) is dissociated and a cDNA fragment complementary to the full-length miRNA molecule is synthesized. Thus, a cDNA molecule of sufficient length is obtained and both ends of this molecule have sites complementary to the detected miRNA, which allows the use of two miRNA-specific primers for PCR. This technological feature provides a high specificity of the method, but its sensitivity is limited. A long RT primer with two complementary miRNA sites can cause a false-positive signal after repeated PCR cycles.
ПЦР реакция, проводимая с наборами ALMIR

Ready to Use Reagents for PCR

PCR enzyme

HS-Taq DNA polymerase, mixture of Tris-HCl (pH 8.6), MgCl, KCl, dNTP mixture, Tween20.

PCR bufer

Contains primers for PCR and miRNA specific probe with a dye label (FAM) and quencher (BHQ1).

Already-existing kits

miRNA RT-PCR Kits «AL-miR-RT-qPCR/hsa-*****»

Kits ALMIR available for order

MoleculeProd. code
mir-106b-5pAL106b-5p
mir-126–3pAL126-3p
mir-145–5pAL145-5p
mir-196b-5pAL196b-5p
mir-10b-5pAL10b-5p
mir-31–5pAL31-5p
mir-221–3pAL221-3p
mir-200b-3pAL200b-3p
mir-200c-3pAL200c-3p
mir-10a-5pAL10a-5p
mir-195–5pAL195-5p
mir-181b-5pAL181b-5p
mir-191–5pAL191-5p
mir-1246–5pAL1246-5p
mir-375–3pAL375-3p
mir-16–5pAL16-5p
mir-29b-3pAL29b-3p
mir-21–5pAL21-5p
mir-451a-5pAL451a-5p
mir-182–5pAL182-5p
mir-205–5pAL205-5p
mir-30a-5pAL30a-5p
mir-371–3pAL371-3p
mir-371–5pAL371-5p
mir-200a-3pAL371-3p
mir-185–5pAL185-5p

Kits ALMIR available
on request

MoleculeProd. code
mir-20a-3pAL20a-3p
mir-34a-3pAL34a-3p
mir-126–5pAL126-5p
mir-145–3pAL145-3р
mir-196b-3pAL196b-3p
mir-31–3pAL31-3p
mir-10b-3pAL10b-3p
mir-221–5pAL221-5p
mir-200c-5pAL200c-5p
mir-10a-3pAL10a-3p
mir-200b-5pAL200b-5p
mir-195–3pAL195-3p
mir-181b-3pAL181b-3p
mir-191–3pAL191-3p
mir-24–3pAL24-3p
mir-24–5pAL24-5p
mir-23a-3pAL23a-3p
mir-23a-5pAL23a-5p
mir-143–3pAL143-3p
mir-143–5pAL143-5p
SnRNA-U6ALSnRNA-U6
SNORD48ALSNORD48
SNORD44ALSNORD44
mir-1246–3pAL1246-3p
mir-375–5pAL375-5p
mir-16–3pAL16-3p
mir-29b-5pAL29b-5p
mir-21–3pAL21-3p
mir-146b-3pAL146b-3p
mir-146b-5pAL146b-5p
mir-182–3pAL182-3p
mir-451a-3pAL451a-3p
mir-205–3pAL205-3p
mir-30a-3pAL30a-3p
mir-200a-5pAL371-5p
mir-93–3pAL93-3p
mir-93–5pAL93-5p
mir-185–3pAL185-3p