RNA is a biological macromolecule with many different functions. Messenger RNA (mRNA) is obtained by transcription of DNA and used as a template for protein synthesis. Protein synthesis is accomplished by ribosomes, which are composed of ribosomal RNA (rRNA) and proteins. Amino acids for protein synthesis are delivered to ribosomes by transporting RNA (tRNA). RNA molecules are also part of the riboprotein involved in the RNA transport process. Non-coding RNA is also very important. They are functional RNA molecules that are not translated into proteins. Such RNA molecules include tRNA, rRNA, nucleolar small RNA (snoRNA), microRNA (miRNA), small interfering RNA, and piwi-interacting RNA (piRNA). They usually play a role in the regulation of gene expression.

miRNAs are a class of endogenous (naturally occurring) non-coding RNAs of approximately 18–24 nucleotides that play a role in post-transcriptional gene regulation. A miRNA may have more than 105 copies per cell, but from the perspective of total RNA quality in each cell, these RNAs are insignificant. Because they are very short, special separation and analysis protocols are often required.

In a typical fast-growing mammalian cell culture, each cell contains approximately 10–30 pg of RNA, whereas in a fully differentiated primary cell, the amount of RNA is much less—about RNA in each cell. The content is less than 1 pg. The RNA molecules in the cells are mainly tRNA and rRNA. mRNA accounts for approximately 1–5% of the total RNA in the cell, but the exact amount depends on the cell type and the physiological state of the cell. In an animal cell, there are approximately 360,000 mRNA molecules that make up approximately 12,000 transcripts, a typical transcript of approximately 2 kb in length. Some mRNA molecules account for 3% of total mRNA, while other mRNA molecules are less than 0.01%. These "rare" or "low abundance" mRNA molecules have only 5-15 copies per cell. However, these rare mRNAs are about 11,000, accounting for 45% of the mRNA.

The genes in an organism are relatively fixed, so the composition of the mRNA represents the way the gene is expressed under given conditions. Analysis of RNA using hybridization techniques, including Northern blotting and microarray analysis, or RT-PCR, transcript sequencing (RNA-seq), can fully reflect gene expression profiles in an organism. However, RNA is relatively unstable compared to DNA. This is largely due to the presence of RNases that degrade RNA molecules.

Ribonuclease is very stable, does not require cofactors, has high catalytic efficiency at very low concentrations, and is not easily deactivated. Ribonuclease contamination can come from human skin and dust particles carrying bacteria and mold. Therefore, the separation and analysis of RNA requires special techniques.

* The content may vary depending on the species, developmental stage and growth conditions.

Small RNAs (miRNAs) are a class of endogenous (naturally occurring), non-coding RNA molecules of approximately 22 nucleotides that are involved in post-transcriptional gene regulation. They have similar characteristics to siRNA molecules.

miRNA molecules play an important role in many biological processes, including cell differentiation and development, cell signal transduction, and response to infection. A large body of evidence shows that the disorder of miRNA expression is the cause and sign of some diseases, including a variety of cancers. Cell-free miRNA molecules can be detected in serum and plasma, and diseases cause their expression levels to change. These findings make the expression of cell-free miRNAs likely to be biomarkers in disease diagnosis and prevention.

Both miRNA and siRNA pathways are associated with double-stranded RNA, but the sources of these RNA molecules are different. Unlike double-stranded RNA that induces RNA interference, miRNAs are encoded by the genome. In addition, the precursor of the miRNA (pre-miRNA) is not completely double-stranded, but a hairpin structure containing a double-stranded region. Unlike RNAi (reference RNAi), the role of miRNAs is primarily to regulate the cells' own genes. Humans have more than 2,000 miRNA molecules, and it is estimated that they regulate more than two-thirds of human genes.

The miRNA system is an endogenous mechanism that regulates gene expression. Mature miRNA molecules regulate the expression of endogenous genes mainly through translational inhibition. In addition, miRNAs can destroy mRNA by rapid deadenylation and removal of caps. The binding site of a naturally occurring miRNA molecule, usually in the untranslated region at the 3' end of the target mRNA. In animal miRNA molecules, partial matching of sequences poses difficulties in determining the true binding site and reduces the accuracy of binding site determination.

miRNA mimics are chemically synthesized, double-stranded RNA molecules (usually 18–24 nucleotides in length) that are transfected into cells to mimic mature endogenous miRNA molecules. miRNA inhibitors are single-stranded (usually 21–25 nucleotides in length), and modified RNA molecules, by transfection into cells, specifically inhibit miRNA function. Mimetics and inhibitors contain some chemical modifications to increase activity or enhance their stability in vivo.

By transfecting miRNA mimics into cells and performing downstream gene expression analysis or phenotypic analysis, the goals and roles of specific miRNA molecules can be elucidated. These experiments can study the biological effects of misregulation of individual miRNAs and can also be used to identify specific target genes for a particular miRNA molecule. After miRNA transfection, if the gene expression level is reduced or increased, the miRNA involved in the study regulates the expression of this gene. Similarly, the role of miRNAs in many pathways can be studied by detecting specific phenotypes after transfection of miRNA mimics or inhibitors.

The effect of miRNA mimic/inhibitor transfection on downstream applications can usually be analyzed by the following protocol:

• A plasmid vector carrying a reporter gene, such as luciferase, and one or more miRNA binding sites located in the 3' untranslated region is used as a target for the miRNA. The mimic/inhibitor of the miRNA is co-transfected into the cell with the vector. After transfection, a reporter gene assay, such as a luciferase assay, is performed. The effects of miRNA mimics and inhibitors were determined by comparing the results of the above transfections with the results of transfection only plasmid vectors.
• The mimetic/inhibitor of the miRNA is transfected into the cell and the expression of the associated endogenous gene (ie, the target of the miRNA molecule under investigation) is detected. The effect of the miRNA mimetic/inhibitor can be determined by comparing the results to the expression of untransfected cells or cells transfected with a negative control. Since miRNAs generally inhibit translation of a target gene without degrading the transcript of the target gene, the expression level of the gene is generally determined by detecting the expression level of the protein, such as by Western blotting. This means that the effects of miRNA mimics/inhibitors are usually not detectable by quantitative real-time PCR.

siRNA

Small interfering RNAs (often referred to as siRNAs) are involved in a variety of biological processes - most commonly RNA interference or RNAi.

Most RNAs are single-stranded, but siRNA consists of two complementary nucleic acid strands, similar to DNA. The siRNA is approximately 20-25 nucleotides in length. siRNAs play an important role in the RNAi process by interfering with the expression of specific genes through complementary nucleotide sequences.

Some approximations of a two-stranded siRNA molecule of 21 nucleotides in length:

• 20 μM siRNA is equivalent to a concentration of approximately 0.25 μg/μl
• The 21-nucleotide siRNA has a molecular weight of approximately 13–15 μg/nmol

RNAi

RNA interference (or RNAi) is a naturally occurring process in cells that shuts down or silences the activity of a particular gene. RNA interference was discovered in 1998 and is now a powerful tool for studying gene function.

RNAi acts by interfering with information carried by messenger RNA (mRNA), thereby inhibiting protein synthesis. When the mRNA loses its activity, the gene is inactivated.

The double-stranded RNA molecule (siRNA) that induces RNAi is cleaved into small fragments by the Dicer enzyme after entering the cell. These small fragments, as a guide, direct the siRNA to bind to mRNA with complementary sequences. The mRNA in these cells is then cleaved, effectively destroying the information they carry and silencing the expression of the corresponding gene.

The process of RNAi is quite complicated. Double-stranded RNA is recognized by RNase III and then cleaved into siRNA molecules of 21–23 nucleotides in size. These siRNA molecules are involved in the formation of RNAi target complexes called RISC (RNA-induced silencing complex), which are capable of disrupting mRNA molecules homologous to siRNA. The target mRNA molecule is cleaved at the center of its complementary region to the siRNA molecule sequence, which then causes the target mRNA molecule to be degraded, reducing protein expression.

Using siRNA

The siRNA molecule is the main effector in the RNAi process and can be synthesized in vitro by chemical or enzymatic methods. The sequence design of siRNAs is critical for efficient gene silencing, and their design approach was developed based on an understanding of the RNAi process and an understanding of the function of naturally occurring siRNA molecules.

Delivery of siRNA is critical for gene silencing experiments. Synthetic siRNA molecules can be delivered to cells by electroporation or lipophilic reagents. However, both methods are temporary. The plasmid system can be used to express short hairpin RNA (shRNA) molecules, which are substrates for the Dicer enzyme and which mature into siRNA molecules in vivo. Such a system can stably inhibit the expression of a target gene. There are also viral delivery systems that are capable of delivering shRNA to cell lines that are difficult to transfect.

RNAi experiment principle and process

The above information is from QIAGEN's official website: http://