Pseudomonas Aeruginosa Gene Expression

Pseudomonas Aeruginosa Gene ExpressionPseudomonas aeruginosa is the gram negative rod shaped bacterium is found in only natural and man made environments. the organism is so stubburn so that it can develop in any environment and alike at high temperatures such as 420C. The unique ability of the organism is categorying biofilm. The biofilms ar formed by the organism with the association with a surface. The surface whitethorn be anything such as cloth, paper, glass etc and any artificial surface. It is the opportunistic pathogen which behaves neutral to the median(prenominal) tissues still it infects once the defences of them ar compromised. The maturement of the biofilm starts with the attachment of the let goly moving planktonic cell to a surface. Gener tout ensembley the twitching motility of the P.aeruginosa helps to use its unipolar flagellum to cohere a surface. The bacterium develops colonies known as microcolonies and adhere to the surface strongly which atomic s um 18 known as pili structures (Baron S et al.,1996). During the biofilm development several factors plays a key role mainly the exopolymeric matrix. The EPS matrix is non visualised instantaneously, but it can be done by exploitation some fluorescent dyes. The functioning of the EPS matrix in holding cells of Pseudomonas aeruginosa unneurotic and forming the mature biofilms. The biofilm develops with cell division and recruitment. The cells interacts via quorum sensing and these interactions atomic number 18 studied under scanning electron micros feign. The biofilm establishment takes place in stages and many changes are discover in its size and shape during the development. The EPS matrix is made up of polysaccharides, proteins and nucleic acids. The exopolysaccharide matrix is the important destiny of the biofilm. The exopolysaccharide brokers such as pilB, pslA, lecA, ureB and alginate genes are responsible for the formation of genus Pseudomonas biofilms. The locus of t he psl polysacchide genes encode the cells to adhere to a surface and thus maintaining the biofilm structure.In this research project we try to quantify the gene view of the above genes pilB, pslA, lecA, ureB using rpoD as the house keeping gene. The biofilms are grow invitro on microcarriers. The microcarriers made up of dextrons in the range of 60-90m in size are employ as a surface for Pseudomonas aeruginosa to form biofilms. The microcarriers are spherical in size where the bacterium can easily adhere on surface. In this array work we try to grow planktonic cultures and biofilms cultures on the microcarriers in a fermentor separately. The genes responsible for the biofilm formation are quantified by comparing the planktonic and biofilm models. The planktonic cells grow precise rapidly whereas the bacterial adhering takes some time to form biofilm in other case. The samples are put in at regular intervals and mribonucleic acid is extracted. The gene human face is quantifi ed using real time PCR. The relative quantification rule of qPCR or real time PCR method does not require any touchstone veer for the compend where the samples are normalised by using an internal run into. The threshold CT values obtained from the curve are utilize to calculate relative quantification by comparative CT method. The results from the planktonic and biofilm gene typeface results are compared. On the other hand we also assay the nutrients such as glucose, ammonia, phosphorus and protease during the fermentation. The submerging levels of the nutrients are also compared for both biofilm and planktonic fermentation.Review of literaturePseudomonas aeruginosa is a member of gamma proteobacteria class of bacteria. The bacteria is gram negative, rod shaped and aerobic belongs to the bacterial family of pseudomonadanceae. The recent developments in the taxonomy based on the 16s ribosomal RNA or conserved macro corpuscles, the family members are divided into eight group s. Pseudomonas aeruginosa is a species chip in in this group sharing the with other twelve members.Pseudomonas aeruginosa is a free-living bacterium, commonly found in soil, water, skin flora and in all(a) man made environments. The organism regularly occurs on the surfaces of the plants and animals. Pseudomonas aeruginosa is an occasional pathogen to plants. It is also an increasingly emerging pathogen of clinical relevance. The organism not thrives in the normal atmosphere but also in the places with little oxygen and thus forms the colonies in natural and artificial environments.Pseudomonas aeruginosa is an opportunistic pathogen for humans, which means that it exploits some publish in the host defences to initiate an infection. The bacteria never infects the uncompromised tissues, but it can infect any if the tissue defences are comprised. It can cause infections such as urinary tract, respiratory system infection, soft tissue infection dermatitis, go down ontro intestinal in fections. It causes a variety of systemic infections in patients particularly with severe burns and repellent suppressed in cases of cancer, AIDS, cystic fibrosis in lungs.CharacteristicsPseudomonas aeruginosa is rod shaped gram negative bacteria. The size of it ranges between 0.5 to 0.8 m by 1.5 to 3.0 m. almost all the strains of Pseudomonas are motile with a unipolar flagellum. It is ubiquitous in soil ,water and on the surfaces in contact with water or soil. It has respiratory metamorphosis and can grow in the absence of oxygen if NO3 is accessible as respiratory acceptor for electron.Generally the bacterium is found in nature in the form of biofilm attached to a surface or substrate, or in the planktonic form as a unicellular organism swimming actively with its unipolar flagellum. It is one of the most vigorous and fast swimming organism found in pond waters. It can grow at temperatures at 42OC or high, but the optimum temperature is 30 to 37OC. it is resistible to a wide v ariety of physical conditions such as temperature and can even resist the salts, dyes, antiseptics and most commonly used antibiotics.Pseudomonas aeruginosa produces different types of colonies from the source they have isolated. They appear like weeny and rough colonies. Moreover the samples collected from the clinical sources look like a hot up egg like structure which is smooth, large with elevated appearance. The other type of colonies obtained from the respiratory and urinary tract secretions looks like mucoid.this mucoid appearance is attributes for the production of alginate slime. These smooth, mucose colonies presumably plays an important part in the colonization and virulence.Pseudomonas aeruginosa produces two types of soluble pigments. Pyocyanin which is a blue-green and a yellow-green and fluorescent pigment called as pyoverdin. Pyocyanin is produced abundantly in the media with low levels of iron and also functions for the iron metabolism of the organism. Many strai ns of Pseudomonas aeruginosa produces pyocyanin but not all the strains does it. Although the organism is classified as the aerobic, sometimes the organism is considered as facultative anaerobe where it can adapt and proliferate the conditions with less or total O2 depletion. The organism can grow in the absence of oxygen if NO3 is available as respiratory acceptor for electron. It can ferment arginine by substrate level phosphorylation where the layers of alginate present surrounding the mucoid cells of the bacteria controls the diffusion of oxygen(Collins FM, 1955), this is witnessed in the cystic fibrosis of the lung infection. Pseudomonas aeruginosa which is found in the biofilm form is responsible for a variety of activities involved which are natural and artificial.Resistance to antibioticsPseudomonas aeruginosa is opportunistic pathogen, has a notorious resistance to antibiotics. The organism is very resistance towards many antibiotics due to the potential permeability barrie r provided by the gram negative membrane. Pseudomonas aeruginosa in the biofilm form makes the cells impervious to the concentrations of the therapeutic antibiotic. The organism gets associated with others like actinomycytes, moulds and bacillus because of the natural environment they live. Moreover the organism have antibiotic resistance plasmids R factors and RTFs, the genes are able transferred by conjugation and transduction following the mechanism horizontal gene transfer(HGT) (Todar.K,2008).BiofilmsBiofilms are defined as a matrix enclosed bacterial populations which are adherent to each surfaces or interfaces. Microbial aggregates and floccules and also adherent populations within the pore spaces of the media are spoken in the definition (Costerton J.W, et al 1995).bacterial biofilms came into existence and became remarkable from their first recognition in their ubiquity. The past researches show that profoundly the growth phase of the biofilms is distinct from the planktoni c. In response to the surfaces proximity, bacterial cells alter their phenotypes (Costerton J.W, et al, 1995). The early stages of biofilm formation the bacteria is in stable juxta position with those of same and other species. The micro environmental conditions, cellular juxtapositions and the extra cellular polysaccharide matrix constitute to the development of the bacterial biofilm. Different bacteria responds to the specific environmental conditions with certain growth patterns, and a structurally complex and mature biofilm develops(Hamilton W.A et al, 1987). Physiological cooperation is the major criteria in formative structure and juxta positions establishment to make biofilms adherent for the surfaces. It has been proposed that large suites of genes are expressed differentially regulated.FormationThe formation of the biofilm initially starts with the attachment of the freely floating bacterial cells to a surface. The bacterial colonies adhere to the surface with weak vander wall forces. These colonies can lead themselves to more adhesion called as pili structures (Baron S et al.,1996). The organism adheres more to the available surface by binary program fission, exopolymeric matrix is produced to develop mature biofilms. The matrix produced holds the biofilm together. Biofilm develops with cell division and recruitment, the cell in the biofilm communicate via quorum sensing. We can observe changes in shape and size in the final stages of biofilm formation.Distribution and ubiquity of biofilmsThe general phenotypic strategy of organism is to change in the mode of growth that to fasten on to the altering growth conditions. The cells of Pseudomonas aeruginosa which is a ubiquitous bacterial species respond to all favourable nutrient conditions. The organism adheres to the available surface by binary fission, exopolymeric matrix is produced to develop mature biofilms. The rod shaped bacteria grows in matrix which is enclosed in a sessile growth mode. Th e cells are protected from the biological, antibacterial, chemical agents and adverse environmental conditions. The periodically shed planktonic cells from the biofilm provide and sacrifice the protection to the new fresh habitats can colonize with the biofilm. The adherent biofilm populations has a significant metabolic activities( Fletcher.M, 1986).The extent of biofilm formation is predicted in the particular system is based on the following principlesMetabolically active bacteria show magnetic inclination towards the surfaces for adhesion in all natural environments.In all environmental conditions the extent of biofilm formation is controlled by the available nutrient for cell respiration and exopolysaccharide production.The organic fertiliser nutrients sometimes tend to associate with the surfaces, in such cases the local biofilm is difficult to trigger. This is more in extremely oligotrophical environments, but bacteria normally does not adhere to surfaces in nutrient less environments.Pseudomonas aeruginosa BiofilmsThe communities of microbial bacteria(Pseudomonas aeruginosa) forms a definite structures with the association with surfaces are abundantly available environment. These structures are often called as biofilms which are less susceptible to antimicrobial agents. These biofilms causes chronic infections and very difficult to have. The extracellular polymeric matrix(EPS) are known to holds the biofilm structure and it performs as a matrix. The EPS matrix holds the biofilm cells together and protects them from shear forces in fludic environments. The extra cellular matrix for Pseudomonas aeruginosa forms the biofilm development in different stages. The matrix holds the cells together on the surface and forms a circumstantial structural design resists to many environmental changes. Pseudomonas aeruginosa biofilm develops in a five stage multicellular cycles, the process is initiated by the attachment of planktonic (free) cells to a surface. Th is is followed by the formation of microcolonies. The microcolonies are seeded and gets dispersed where the cells form the microcolonies comes out to occupy a new surface( Ma. L et al, 2009Stoodley.P et al,2002).The EPS of Pseudomonas aeruginosa composed of polysaccharides, proteins and nucleic acids. It is the important component of the biofilms. The exo polysaccharide genes lec A , Pil B, Psl A, alginate are responsible for the formation of Pseudomonas biofilms. The locus of the Psl exopolysaccharide encodes bacterial cells to surface and thus maintaining the biofilm structure. The matrix which is anchored by the Psl is not visualised directly during the developmental stages of Pseudomonas aeruginosa biofilm.In addition to the present scenario of exopolysaccharides, extracellular deoxyribonucleic acid also plays an important part in . P.aeruginosa biofilm matrix (Allensen-Holm M et al, 2006). The extracellular desoxyribonucleic acid is derived from the chromosomes and it function s for signalling between the cells interconnecting the biofilm matrix. In the biofilm matrix, extracellular cellular DNA promotes cation gradients, release of genomic DNA and induces antibiotic resistance ( Mulcahy H, et al. 2008).Real time PCRPolmerase chain reaction or precisely PCR is introduced by Kary mulis in 1980, which really a revolution in the molecular biology. PCR is the standard procedure to amplify small quantities of DNA molecule into large quantities. The method depends on thermal cycling which involves the continuous warmheartednessing and cooling of reaction for DNA melting and enzymatic replication of DNA. PCR applications employ a general heat stable DNA polymerase such as Taq polymerase enzyme isolated from the bacterium Thermus aquaticus.PCR method is involves three major steps.1) Denaturation 2) Annealing 3) Elongation. In the first step DNA templet is denaturated to single strand at high temperature of 940C. In the second step, single stranded DNA template annealed with primers, the temperature is 650C. At 720 C the DNA starts elongation which is threesome step. DNA polymerase synthesizes new complementary strand to the template DNA strand.Real time polymerase chain reaction or qualitative polymerase chain reaction is a laboratory technique which not only amplifies but also quantifies the marking DNA molecule. it helps the user for detection and quantification(absolute and relative) of many specific genes in a present in a given DNA sample. The general principle of polymerase chain reaction (PCR) is also used in the RT PCR. Unlike PCR, RT PCR is a new engineering product is spy at the end of the reaction.There are two common methods for the product detection in RT PCR1) Non -specific fluorescent dyes which interacts with the double stranded DNA molecule2) Sequence specific DNA probes consists of oligonucleotides that are fluorescently labelled with reporter. The product is detected after the hybridization of the probe and complemen tary strand.The RT PCR is used to amplify the reversly canned cDNA products from the mRNA, which is becoming the effective and powerful tool in the field of molecular biology to quantify and study the gene expression. The RT PCR method is very scant(p) to apply provides the reliable, accuracy and rapid gene expression quantification. The accurate nucleic acid quantification requires mathematical application for data analysis(Michael W.P., 2001). The real time PCR provides the very accurate and duplicable quantification of gene copies (Heid C.A., 1996). Unlike other PCR methods, RT PCR does not require post process sample handling, prevents product contamination, provides faster and accurate assay results. Real time PCR is very accurate and less labour usage than the present gene quantification methods. Nucleic acid sequence quantification has an important role in the biological research. Measuring the gene expression have been extensively used in the biological responses to vario us external stimuli( Tan et al, 1994 Huang et al,1995). Gene and genome quatification also used for HIV demonstrates the change in the expression level of virus in the disease period( Conner et al. 1993) (piatak et al, 1993).There are two types of quantification methods used in real time PCRAbsolute quantificationRelative quantificationAbsolute quantification determines the stimulant copy number generally by relating the PCR signal to a standard curve. In this method equal input measurings of sample, each sample under analysis amplifies with identical efficiency up to the point of qualitative analysis( Heid et al,1996). We quantitate the unknowns based on a known standard quantity. The unknown samples are compared to the known by creating a standard curve. The input copy number of the PCR signal is related to standard curve in absolute quantification method. Sometimes it is not necessary to know the copy number but it is important to calculate the relative change gene expression ( Livak.K.J et al, 2001).The second method is relative quantification, we analyse the changes in gene expression in a given sample relative to another denotation sample(untreated control sample). Relative standard again consists of two types of methods they are relative standard curve and comparative Ct method. In the relative quantification the PCR signal is related to the targeted transcript of the samples in the group. The new applications of comparative Ct method is the 2-Ct method which is efficiently useful for the analysis of qPCR data.The 2-Ct method(Livak.K.J et al , 2001)Derivation of 2-Ct methodThe equation for the amplification of PCR can be written asXn=X0 (1+Ex)n(1)Where Xn is the number of target molecules at cycle n of the reaction.X0 is the initial number opf target molecules.Ex is the efficiency of the target amplicon.n is the number of cyclesCT is the threshold cycle, indicates the halfway cycle at which the amount of amplified target reaches a fixed threshold.XT =X0 (1+Ex)CT.X=Kx(2)Where XT is the threshold number of target molecules.CTX is the threshold cycle for target molecule and Kx is constant.A similar reaction for the internal control gene isRT =R0 (1+ER)CTR=KR-(3)Where RT is the threshold number of part moleculesR0 is the initial number of reference moleculesER is the efficiency of the reference molecule.CT.R is the threshold cycle for reference amplicon.KR is a constant.Dividing XT and RT, we get ,(XT/ RT )= (X0 (1+Ex)CT.X) / (R0 (1+ER)CT.R )= (Kx / KR ) = K-(4)For real time amplification using Taqman probes the exact values of the XT and RT depend upon the number of factors including the reporter dye and fluorescence properties of the probes, efficiency and purity of the probe, florescence threshold setting. Thus k cannot be equal to one. Assume the efficiencies of target and the reference as equal.ER = Ex=E(X0 / R0) (1+ E) CT.X- CT.R =K(5)XN (1+E)-CT =K-(6)Where XN is equal to the normalised amount of target gene (X0 / R0) and C T is the difference of the threshold cycles of target and reference (CT.X- CT.R ).The expression can be written asXN=K(1+E) -CT -(7)If we divide the whole equation by XN for any sample q by XN as the calibrator(cb)XN.q /XN.cb = K(1+E) -CTq / K(1+E) -CTcb =(1+E) -CT here(predicate) -CT = -(CTq -CT.cb)For amplicons which are designed less than 150 bp, primer and magnesium concentration are optimised, the efficiency is nearly equal to 1. Therefore the amount of target gene, normalized to an endogenous reference which is relative to a calibrator, taken asAmount of target =2-CT .When the threshold is set for n standard deviations in the speckle , above the mean of the base line emission from cycles 0 to n. The standard deviations is calculated from the data attained from base line of the amplification plot. When the fluorescence exceeds the threshold limit, CT values are determined at that point. The CT values are ten times more than the standard deviation of base line. The point at wh ich amplification plot crosses the threshold can be defined as CT (Heid et al, 1996).Materials and methodsIdentifying pseudomonas aeruginosaGram strainingPrepare a small amount of dirty on a clear glass slide.Heat fix the smear to slide. contribute a drop of crystal violet on to smear allow for 60 seconds.Rinse the slide with water.Add a drop of grams iodine to the smear allow for 60 seconds.Rinse the slide with water.Add 95% absolute ethanol to destain the crystal violet. Rinse again with water.Add a drop of saffronin over the smear, allow for 60 seconds. Rinse with water.Blot dry the slide, do not rub. Observe the smear under microscope.10. If the colour of the bacteria appears pink it is gram negative, it is gram positive if it appears purple. Rod shaped pink coloured bacteria is clearly observed for gram negative pseudomonas aeruginosa.Shake flask methodPrepare media of nutrient farm animal about 13 grams per litre of distilled water. smorgasbord well and pour 50 ml of media into a 250ml conical flask. Plug the conical flask and wrap the mouth with aluminium foil. The less volumes of media in the flask increases surface area and oxygen transfer rate(OTR). The flasks are autoclaved at 1210 C and 1.5 lbs squash for 1 hour 15 minutes.Planktonic-contains 50 ml of nutrient brothBiofilm-contains 25ml of PBS with microcarriers plus 25 ml of nutrient broth.Inoculate a loopful of pseudomonas aeruginosa into 50 ml nutrient broth aseptically. Allow the bacteria to grow overnight. Take the optic density at 550nm using spectrophotometer.The amount of the inoculums to be added to the shake flasks for analysis is calculated using the formula belowAmount of sample to add into shake flask= working concentration X volume(ml) / stock(OD) obtainedAfter inoculation the flasks are kept in the orbital shaker at 300C. The samples of 1 ml is collected for RNA extraction at particular time intervals at 0,1,2, 4,24 and 48 hours for planktonic. In respect the biofilm samples ar e collected at time 0,22,24,26,44,46 and 48 hours as their growth is slower than the freely suspended planktonic cells. Then 2 ml of RNA later issue is added to the collected samples for RNA stabilization. The optical density is regularly monitored to check the growth of pseudomonas aeruginosa .FermentationThe fermentation process we perform here is batch fermentation. The bioreactor used for the study is a small scale fermentor from APPLICON BIOTECHNOLOGIES. The process will take place in a 3L fermentor(consists the working volume of 2.5L) for about 24 hours. During the run the criteria of temperature is kept at 300C . The pH is run free, stirrer speed is set at 600rpm for planktonic culture. The fermentor is run free for 24 hours overnight to check the production of foam. The cold finger helps to control the temperature by feeding water from/to the reactor. The o ring is analyse for any leakage. The sample port is a provide for sampling using back pressure and forces the liquid into a glass recipient. The inlets for acid, alkali and antifoam are linked to durham bottles, going through the pumps. The durham bottles are blank, because above parameters are not controlled in this process. Air is pumped through a filter. The condenser is connected to water that comes out of the fermentor. When the water is warmer than outside, condensed water reaches the condenser. The gas goes from an outlet connected to the condenser.The fade out oxygen probe is connected to dO2 cable from the machine. It measures the amount of dO2 in the media. This probe needs to be kept in the buffer, when not in use. The probe should be plugged for 10 to 12 hours before the inoculation inorder to polarize. The stirrer is placed on the top of fermentor. The vessel is heated with the help of heating pad and temperature is detected using temperature probe. The air inlet is set up through the pumps and its volume is controlled with a rotometer in front of the machine. In this case 2.5L of a ir/min is supplied to the fermentor. For the gas analysis of oxygen and carbon dioxide the gas is passed through a drying column and reaches the monitor which is already set up for calibration. we run two runs of fermentor for both planktonic and biofilm cultures. The parameters such as temperature, aeration for planktonic and biofilm cultures are almost same. The only parameter varies for both is stirring is maintained at 600rpm for planktonic and 200rpm for biofilm fermentation.ProcedureThe fermentor is sterilised and all the valves are sealed with foil before going into the autoclave. The fermentor is run for about 24 hours before adding the inoculum to check any formation of foam. About 20 ml of an overnight culture is added to the fermentor.Sampling About 7ml of sample is collected for every 20min for planktonic and every 2 hours for biofilm until the exponential phase is reached. The sample collected is preserved in the freezer for different analysis. The sample is assayed for gene expression by adding 2 ml of RNA later resolving for RNA stability. The samples are assayed for ammonia, phosphorus, glucose and protease. The fermentation conditions are listed below.RNA EXTRACTION (protocol provided by applied biosystems)Materials required2-mercapto ethanol, ampere-second% ethanol, 10% SDS(in RNase free water), 0.5 l/sample.Lysozyme solution100 l/ sample10mM Tris HCl (PH 8.0)mM EDTA10mg/ml lysozyme (in RNase free water)Lysis bufferBefore beginning the lysis and homogenization steps, prepare a fresh amount of lysis buffer containing 1% 2-mercapto ethanol for each purification procedure. Add 10l 2-mercaptoethanol for each 1ml lysis buffer. Use 350 l of freshly alert lysis buffer for 1109 bacterial cells.Lysis and homogenization product 1109 bacterial cells and transfer them to an appropriately sized microcentrifuge tubes. Centrifuge at 500xg for 5 minutes at 40C to pellet cells. thresh the supernatant. Add 100 l of prepared lysozyme solution to the cell pel let and resuspend by vortexing. Add 0.5 l 10% SDS solution vortex to mix well. Incubate the cells in the tube for 5 minutes at room temperature. Add 350 l lysis buffer prepared with 2-mercaptoethanol. Vortex to mix well. Transfer the lysate to a homogenizer inserted in an RNase free tube and centrifuge at 12,000xg for 2 minutes at room temperature. Remove the homogenizer when done.Purifying the RNA from bacterial cellsThese steps are followed to bind wake and elute the RNA from the P.aeruginosa cells sample.Add 250 l 100% ethanol to each volume of bacterial cell homogenate.Mix thoroughly by vortexing to dispersing any perceptible precipitate that may form after adding ethanol.Transfer the sample(including any remaining precipitate) to a plait pickup arm( with a collection tube.Centrifuge both the spin cartridge and collection tube at 12,000xg for 15 seconds at room temperature. Discard the flow through and re-insert the spin cartridge in the same collection tube.Add 700 l of was h buffer I to the spin cartridge. Centrifuge at 12,000xg for 15 seconds at room temperature. Discard the flow through and the collection tube. piazza the spin cartridge into a new collection tube.Add 500 l wash buffer II with ethanol to the spin cartridge.Centrifuge at 12,000Xg for 15 seconds at room temperature. Discard the flow through and re insert the spin cartridge into the same collection tube. The steps are iterate once again.Centrifuge the spin cartridge and collection tube at 12,000Xg for 1 minute at room temperature to dry the membrane with attached RNA. Discard the collection tube and insert the spin cartridge into a recovery tube.Add 50 l of RNase free water to the centre of spin cartridge.Incubate at room temperature for 1 minute.Centrifuge the spin cartridge and recovery tube for 2 minutes at 12,000Xg at room temperature. gillyflower the purified RNA or proceeded for DNase treatment after RNA treatment.(store purified RNA at -800C for long term).Agarose gel electroph oresisThe RNA samples after extraction are checked for the RNA signal. To visualise the RNA 1% agarose gel electrophoresis. The materials required for agarose gel electrophoresis is TAE buffer and agarose.TAE buffer(50x Tris acetate)242 g of Tris base57.1 ml glaciel acetic acid100 ml of EDTA 0.5M PH .8.0700ml of dH2O .About 20 ml of above solution is mixed with 980ml of distilled water to make it 1X. 1 gram of agarose is added to 100 ml of TAE buffer. The solution is mixed well. The solution is heated until the agarose is completely dissolved and solution becomes colourless. About 2.5 l of ethidium bromide is added to the solution to visualise the RNA bands. Special care is taken while adding the ethidium bromide because it is bio-hazardous. Then about 5 l RNA sample is mixed with 1 l of DNA loading dye. The RNA samples are RUN with the potential difference of 80V with DNA Hyper ladder II as a standard.DNase treatment (protocol and kit provided by applied biosystem)The DNase I treat ment is performed to remove any traces of genomic DNA from the eluted RNA.ProtocolAdd 10x DNase buffer and DNase I to the RNA sample in the following compositionAmount component1/9 th volume 10x DNase buffer4 l DNase IThe components are mixed with gentle pippeting. Then the mixture is incubated at 370C for 90 minutes. DNase inactivation reagent is added to the solution and mixed well, leave at room temperature for 2 minutes. The samples are centrifuged at maximum speed for 1 minute to pellet the DNase inactivation reagent, after that the RNA solution is transferred to a new RNase free tube. The samples are run on 1% agarose gel electrophoresis to check the DNA contamination. We fou