INTRODUCIONFrom at a time, which resulted in

INTRODUCIONFrom the last 15 years, global analysis of mRNA or of gene has been emerging as important strategy for biological discoveries. A number of powerful methodologies such as bioinformatics, robotics, parallel processing and high throughput methods have been devised in order to analyze the whole genome. Globally gene expression analysis in prokaryotic cell is insulated under the eukaryotes due to the lacking of polyadenylation of prokaryotic mRNA, which then have selective labelling on mRNA in already presence of large amount of transfer RNA and ribosomal RNA. Population of RNA in a cell is highly unique and complex to measure. It ranges from a few to several thousands in different species and also differ in size. Knowing the number of RNA molecules, shows the interest to study the expression pattern of RNA in target cell or issues.In previous days the methods to study the gene regulation and gene expression allow the scientists to focus only on one gene at a time, which resulted in the loss of many biological events. From the last 10 years’ abundance of methods have established which allow the scientists to examine mRNA expression levels of thousands of genes in only one experiment. Up till now a number of RNA expression analysis methods have been devised such as RNA sequencing, Microarray, Northern blotting, EST (expressed sequence tag) and many others. Each of these methods have its own advantages and disadvantages.4Central Dogma In living organisms cells are continually in a process of activating or deactivating genes by gene expression which have the ability to synthesize particular protein through protein synthesis. A particular gene code for a particular protein so when there is a need of a particular protein that gene is activated which codes for that protein. For the synthesis of protein, first of all an RNA copy of gene’s DNA is produced. This RNA copy is messenger RNA.The quantity of mRNA produced is directly equal to the amount of synthesized protein so therefore, by quantifying mRNA amount we are able to get the amount of protein. We usually measure mRNA quantity because it is quite easy to detect mRNA amount as compared to protein synthesized. Gene activation levels vary between healthy cells and carcinogenic cells.Application of RNA expression analysisSome of the techniques to analyze the RNA expression are:1. Northern BlottingWhen a gene is identified, first of all we determine the mRNA size and alternative splice variants. In this way we can evaluate size of protein and DNA sequencing data. Therefore, we use the method of northern blotting to analyze RNA. This was the first ever technique to quantify the RNA. It is used to visualize the differences in quantity of RNA produce by different genes at different times.PrincipleIn the first step of northern blotting we isolate pure and intact RNA from cell or tissue of interest. The isolated RNA either total or polyA mRNA is separated on the basis of their size5by the process of gel electrophoresis. The size based separated mRNA are then moved to a solid support filter membrane, which is hybridized by little amount of 32S or 32P.radioactively labeled short fragments of DNA or RNA probes. They bind correspondingly to the gene of interest. These probes form a complex with RNA where they find their complementary sequences. These RNA-probe hybrids can be detected by radionuclides. The emitted signals of the probes indicate the amount of target RNA.DetectionThe RNA-probe target hybrids or complexes are then detected by the help of ribonuclide. The emitted signals tells about the amount of RNA quantified. These filters are then exposed tothe X-ray films, this film is exposed by radioactivity of the probe and then bands are visualized. Thus the band’s intensity tells us how much RNA was there in the sample which directly indicates how strongly the gene of interest is expressed.QuantificationWe also do relative quantification of gene expression by comparing two or more signals from cell or tissue levels. However, by using standard curve of known sample in vitro6corresponding to target RNA, we do absolute quantification. To get perfect result, housekeeping genes are used which avoid unfair differences due to unequal RNA transfer to membrane.Advantages of northern blotting? Northern blotting is the very first technique to quantify single mRNA sample.? It gives enough data about tissue and developmental levels during gene expression.? It has good control quantitative ability and determine the length of mRNALimitationsTypically, only a single gene is analyzing and quantified at a time by using northern blotting technique. Secondly there occur variations in the quantification comparisons from one blot to another blot majorly at large scale. We need multiple blots and large amount of RNA and other reagents for the analyzing and quantifying more than 10-20 RNA samples- which accounts in large cost and more time-consuming. It gives low throughput.2. Real time PCRIn order to quantify the differences in mRNA expression, real time PCR was developed. PCR are more sensitive and dynamic because they involve amplification steps and they require particularly low amounts of RNA. It analyzes expression of even single transcript in RNA obtained from a single cell.PrincipleFirst of all, mRNA is isolated and purified. As RNA contains virus PCR doesn’t work on RNA templates therefore reverse transcription occurs in which we make cDNA copies of RNA sequences by using gene-specific primers. This cDNA is then used as a template for qPCR reactions. Mostly this reaction occurs in 96 well-plates reaction mixture.At the end final product amplicon is subjected to run on gel and probed.One-step Vs. Two-step RT-Qpcr7Real time PCR can be performed in two ways. In one-step reaction, all the buffer, reverse transcription and PCR steps are combined in a single tube by using reverse transcription enzyme and DNA polymerase. It also use sequence-specific primers. However, in two-step process the PCR steps and the reverse transcription occurs in individual tube separately having different buffers, primers and reaction conditions.However, in two-step assay three different primers can be added which include oligodT, sequence specific or random primers but most commonly oligodT and Rndom primers are used. They start to anneal with template strand and provide reverse transcriptase enzyme for synthesis.Real-time PCR- A kinetic approachReal- time PCR is a kinetic process because it can monitor the concentration of PCR product at early stage when it is still linear as opposed to endpoint detection. In other words, fluorescence intensity is measured every cycle. However, in conventional PCR we cannot detect amplicon concentration within the reaction, only we can measure at the end of reaction i.e. only electrophorese the final product not reflecting starting amount.8Detection of ampliconReal-time PCR has the ability to detect the fluorescence emitted by using a fluorophore-conjugated hybridized probe when complexed with dsDNA, which indicates concentration of amplicon production when excited. The emitted light has the signal strength which is directly proportional to the amount of PCR product.SYBR green is a dye which can be used to detect amplicon, it has the ability to bind to dsDNA and fluoresce brightly hence detect synthesis of DNA during reaction. SYBR green has advantage over ethidium bromide because first it gives more fluorescence secondly it’s ratio of fluorescence is higher than ETBR.Advantages of Real-time PCR? No specific amplification occurs during reaction and is not more expensive.? It gives high throughput and lowcontamination and cause high RNA quantification? It has the ability of ultra-rapid cycling and has wider dynamic range.Disadvantages of Real-time PCR? It requires high technical skills and support and cause high equipment cost.? It has small RNA stability and have inter and intra-assay variation? It causes DNA contamination during mRNA analysis.3. MICRO ARRAYMicroarray or the gene chip is being the most famous method for the scientist to analyze the gene or RNA expression. It is a rapid, reliable but somewhat difficult method with high reproducibility and high quantitative approach to study the expression levels of thousands of genes. This method can also be used to compare the two different samples, about the difference of gene expression in two genes. Microarray is basically a method to make spotted array of thousands of different oligonucleotides or c DNA, corresponding to thousands of different genes.PrincipleIn order to analyze the RNA, it would first start up with RNA sample, for this purpose a series of event took place. Biochemical reaction is done to generate the fluorescently labelled cRNA, OR ss-cDNA probe. Probe is a mixture of known sequences of cRNA or DNA. It can9either be attached to nitrocellulosic membrane(solid) by robotic means, where a needle applies the cRNA to the plate, or by a method similar to making silicon chips for computers and so called gene chip. It is then hybridized to microarray and scanned with laser. Then the RNA sample which were fluorescently labelled with 32 p dyes are added to the spotted array and allow the hybridization. Hybridization refers to the annealing of two nucleic acids to bind. The expression level is the measured by measuring the intensity of bound probe to each spot. At least two samples must be hybridized to chip to be analyzed. Then nonspecific binding is washed off and the hybridization are scanned with the help of scanner.DetectionA scanner is used to detect the expression levels, it consists of three parts, a laser which causes the hybrid bonds to fluoresce, a camera which records the images produced when the laser scans the plate and the computer that allows us to immediately view our results and it also stores our data. Those genes which were fully expressed are regrouped and are present in the form of large dark band and those with least expression are just shown as spot.Types of microarray1. Oligo nucleotide microarrayBasically RNA expression analysis is done with oligonucleotide microarray. The array is consisting of a 544 × 544 grid of 24 × 24 ?m regions and each contain 10^7 copies of selected 25-mer oligonucleotides) of know sequence. Probe oligonucleotides are organized in pairs, one of which is flawlessly complementary to the target sequence and one with a single base mismatch at the central position, which serves as a control for nonspecific hybridization.10Probe sets are present for 4,403 “b-numbers,” which include all 4,290 predicted ORFs, as well as all rRNAs and tRNAs. The top half of the array contains oligonucleotides targeting ORFs and various untranslated RNAs, and the bottom region targets intergenic regions and the extreme bottom has probes for tRNAs and rRNAs.2. Spotted microarrayIt is developed by Pat Brown in Stanford. It is use to analyze the long PCR products of known genes of almost 100 nucleotides, that are spotted on solid glass.4. RNA SEQUENCINGRNA sequencing is being revolutionizing since past few years to study the transcriptome. It is highly sensitive and most accurate way to measure the RNA expression in entire transcriptome. It enables us to visualize the unseen or undetected changes, occurring in the disease states. It is able to capture almost all the transcript. It also has low background noise and requires less RNA sample. RNA sequencing detect the transcriptome isoforms, gene fusions, allelic specific gene expression and single nucleotide variants. It uses the NGS (next generation sequencing) method to unravel the amount and expression RNA present in cell.11Applications of RNA sequencing? In many fields like clinical and pharmaceutical, there are a lot of applications of RNA expression analysis.? It has ability to detect side effects of drugs and xenobiotic.? It also determines physiological condition of cell and tissue.5. ESTEST is Expressed Sequence Tag, which is used to analyze the genes expressed in particular tissue and partially for the sequencing of transcripts. It provides the largescale analysis of genome.PrincipleEST involves the creation of cDNA libraries, which represents all the expressed mRNA in a cell or tissue. Then sequencing of thousands of cDNA is done and a database is formed which identifies and counts all the genes, which were expressed and naming as expressed sequenced tags. It does not require the existence of transcript to measure its level of expression. A pool of RNA sample is generated by sequencing thousands of EST to identify differentially expressed genes.