MicroBiology

mRNA refers to messenger RNA. The gene expression studies based on the microarrays enable the simultaneous investigation of the relative amount of messenger RNA for a number of genes with the help of the fluorescently labeled nucleic acid targets. The most of the universal methods makes use of the enzymatic techniques like oligo-dT primed reverse transcription for producing the DNA. These labeling methods have found to have some shortcomings such as laborious protocols, sequence bias, introduced labeling, inability to perceive small alterations in the expression levels and very high experiment-to-experiment variability.

In such cases, a novel and an innovative method of labeling is used. This method makes use of the platinum linked cyanine dyes to chemically and directly label the mRNA from a very little amount of the total RNA. The directly labeled mRNA yields unbiased and highly accurate data of the differential gene expression. The gene expression data that is perceived by using the mRNA labeling method is highly precise without any errors at all. There is no labeling bias and we see a dynamic range over a number of orders of magnitude. This method boasts the highest accuracy levels in identifying the differentially expressed genes. It also cuts down the requirement to run several replicate assays. Thus, minute changes in the expression of genes is now possible to get detected on a large-scale gene expression profile assays with the help of such an easier, quicker as well as simpler procedure.

Nevertheless, the use of the random primers may outwit some of these issues along with the enzymatic labeling techniques, but still we can say that the mRNA Labeling system is quite beneficial. It is an innovative labeling technique that makes use of chemical reagent to label the mRNA directly with the fluorescent dyes. This mRNA is labeled in the entire RNA mixture in a single step non-enzymatic reaction; thereby reducing the errors that have been occurred in the previous methods because of multiple steps. The mRNA thus formed, is free from any labeling bias and it produces reproducible expression profile of the gene and also enables a great amount of accuracy in identifying the differentially expressed genes.

mRNA Labeling System- there are some reagents to be used to prepare the first strand of the DNA for microarray Synthesis. These reagents are highly efficient and full length. Random primers and Oligo dT are used for DNA synthesis. Adequate reagents are provided for ten labeling reactions and they are supplied with the control templates for the Quality Check purpose. This mRNA Labeling system provides the reagents for reverse transcription and for labeling the cDNA from the mRNA templates. The labeled cDNA is, thus used for northern blot applications, FISH or as probes for the microarrays. This mRNA labeling system is stored at a temperature of about 20 degrees C and it remains stable for atleast 6 months.

RNA
RNA stands for ribonucleic acid. It is a nucleic acid that comprises of a big chain of the nucleotide units. Each nucleotide has a ribose sugar, a phosphate and a nitrogenous base. Ribonucleic acid (RNA) is similar to the deoxyribonucleic acid (DNA) to a great extent. The only difference lies in some of the structural details. DNA is double stranded and RNA is single stranded; DNA nucleotides contain deoxyribose and RNA contains only ribose; DNA has the base thymine instead of uracil as present in RNA.

There are generally three types of RNA. These three types include Messenger RNA (mRNA), Ribosomal RNA (rRNA) and Transfer RNA (tRNA). Now let us have a brief overview on each of these types of RNA.

Messenger RNA- mRNA is the replica of the data that is being transmitted on DNA by the genes. The function of this mRNA is to carry the data that is present in the DNA to the translation mechanisms. The mRNA is usually synthesized from the DNA’s gene segment. This RNA has all the data of the amino acids in proteins that are to be synthesized on the primary sequence. It transmits the code in the cytoplasm where the synthesis of proteins takes place. The messenger RNA is heterogeneous in sequence as well as structure. Due to this diversity in size and sequence, there is not a specific structure of mRNA determined. It always has a five-inch cap containing the triphosphate linkages. This cap is an identification that the RNA molecule present in the structure is the mRNA.

Ribosomal RNA- the rRNA is a constituent of ribosome’s that are the regarded as the protein synthesis factories in the cells. The rRNA molecules are found profusely in the cell. 80% of the RNA molecules found in the cells are comprised of the rRNA. The rRNA plays different roles in the process of protein synthesis. Firstly, it plays a catalytic role by forming a part of peptidyl transferrase activity. Then, it plays the recognition role by getting occupied in the exact positioning of the tRNA and the mRNA. And finally, it plays the structural role by getting folded into 3-D shapes and forming a gibbet, wherein the assembly of the ribosomal protein takes place.

Transfer RNA- the tRNA is referred as the data adapter molecule. It bridges the gap directly between the DNA information and the sequence of amino acids in proteins. Thus, it can interpret the information contained in the DNA. There are more than twenty various types of tRNA molecules present in the cells all between the range of 75 - 95 NT. The tRNA’s in all the organisms features a similar structure and orientation. It has three loops and four arms and sometimes, it has an extra loop. The synthesis of the tRNA takes place in two parts. The tRNA body is copied from the tRNA gene. All the tRNA molecules share the same type of acceptor stems and this is added after the synthesis of the body. This is generally replaced at the lifetime of the tRNA molecule.

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MicroRNA is also abbreviated as miRNA. It is a single strand of RNA molecules that is upto 21-23 nucleotides in length. It is responsible for regulating the expression of genes. The genes transcribed from DNA encode the miRNA. The mature miRNA molecules are complimentary to some of the messenger RNA to some extent. They perform the important function of down regulating the expression of genes.

MiRNA is attached to a small piece of mRNA that is the master model to build the proteins in a non-coding pattern at one end of the molecules. These micro RNA’s are very small molecules that are coded in the genomes of animals and plants. They are highly conserved and play the role of regulating the gene expression by connecting the untranslated areas of particular mRNA’s.

Generally, the miRNA’s are transliterated as a part of the long RNA molecule. About 60% of this micro RNA’s are expressed independently; whereas the 15% of it in clusters and remaining 25% are expressed in introns. The mammalian genomes can encode about 200-500 miRNA’s that can together control the expression of 1/3^rd of all the genes.

The micro RNAs are produced and they function in the tissues, cells and organisms. They regulate multiple genes and are present in the higher Eukaryotes. Thus, the miRNA has adequate potential regulatory circuitry. They are the key regulators of the processes including cell death, cell proliferation, cell differentiation, fat metabolism, and apoptosis. The recent studies reveal that the expression of the miRNA is associated with the brain development, viral infection, colonic adenocarcinoma chronic lymphocytic leukemia and Burkitt’s lymphoma.

MiRNA and Cancer- it has been found that there are some miRNA’s that have a link to some type of caner. The patterns of gene activity distinguishing the kind of cancer can be detected by measuring the activity between the 217 genes that encode the miRNA. The signatures of miRNA make it possible to classify the types of cancer. This helps the doctor in determining the original type of tissue causing cancer and thus, the treatment course can be targeted based on the original type of tissue. The profiling of the micro RNA has enabled in verifying the patients having chronic leukemia with slow or aggressive forms of cancer.

MiRNA and Heart Disease- miRNA plays an important role in the functioning of the heart and its role during its development is quite significant. Studies show that the level of expression of a particular miRNA alters in diseased human hearts. It has distinct roles to play at the time of the development of heart, and pathological functions include regulating the key factors necessary for cardiogenesis, cardiac conductance and hypertrophic growth response.

The miRNA’s are significant for the development of the organisms and are expressed differentially in the tissues. They are involved in the processes of viral infection and are also associated with oncogenesis. The activity of these miRNA’s can be blocked experimentally using a Morpholino Oligo, locked nucleic acid or methyl RNA Oligo. The steps in its maturation process can also be blocked using steric blocking oligos.

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