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Differential Expression Pattern

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Welcome back to the course of, Plant Developmental Biology.So, in last class we were discussing, reverse genetics based approaches, which we are being,which we are taking for, to study the developmental.So, in that class we have covered identification and isolation of genes for developmental functions.Now, we started the functional genomics based approaches.So, in functional genomics based approaches, what we discussed the first step was monitoringgene expression.And now, so we have seen how to check the expression pattern of individual genes.Now, we will mostly focus on the global genes.How to study the expression pattern of the Global genes?And what are the way how to study the expression pattern?The important thing here is that, you can again fix the problem.So, the main goal of a genomics or Global gene expression analysis is, to identify thedifferential genes in different conditions.It can be different organs, it can be different tissues, it can be different stages, it canbe different conditions between a wild type and mutants any combination.Whatever you can choose with respect to the development and then you take the genomicsbased approaches and identify all the genes which are differentially expressed in betweentwo conditions two organs or whatever.And there are many way to study a differential gene expression.So, I will list some of them here, but we will focus on only few.So, these were the way to study.First was the serial analysis of gene expression, SAGE.Here people started by obtaining some short sequence tags from a large number of cDNAclones.And they, then they were sequencing it and identifying the differential gene expressionpattern.Then another way was differential display RT PCR, here what people used to do, theyused to take a two population of total RNA, they used to make cDNA.They used to run a gel and then try to identify which particular band is missing in one conditionand present in another condition or vice versa.And then they used to cut that band, amplify the fragment, clone, sequence and identifywhat are the genes.Another way of doing was the subtractive hybridization, this was PCR based amplification of only cDNAfragment that differs between two conditions.And so, basically here what used to do, there were two population of the messenger RNA,you make cDNA then do the subtraction, subtract.After subtraction, you identify or you isolate only those populations which are differentiallyexpressed and then specifically you can amplify them and then identify them.Then the technique, this technique was very widely used in most of the genomics basedexpression, differential expression pattern, analysis which was microarray.Microarray was the technique, where small fragment or small oligo single stranded oligoswere spotted on a chip and then the total RNA maybe was, isolated from a particularcondition to identify the differential expression.We will see micro RNA microarray in great detail, how this has been used.And this is one of the most used technology in the plant developmental biology.And now recently next generation sequencing has, actually changed the entire scenario.Now we are doing lot of RNA sequencing and this has lot of benefits over the microarray.So, these are the different methods which is which are being used to study the differentialexpression pattern in different conditions or different developmental processes.So, I before going to the micro RNA and RNA sequencing in detail, I would give some ofthe typical examples of different technologies, which was used.For example, if you look this, this research here the transcript profiling was done inrice seedling using SAGE as a method.So, this was used and people have identified the differential expression here.Then this is one example, where differential display RT PCR was done.So, this is MADS box containing transcription factor in rice.And what was important here that, this gene which is MADS 1, it was expressed in sepalequivalent.Which in case of rice, is lemma and palea, but it was not expressed in the inner organ.But when the gene was mutated, when this gene was silenced, the effect was also seen inthe inner organs, inner floral organs for example, lodicules, stamens.Then this was the question that, if the gene is not present in this tissue how it is regulating?So, one of the possibility was that, maybe it is activating because at very early stage,it was expressed throughout the floral primordia.So, it was hypothesized that maybe, it is activating some early regulators at very earlystage, whose expression is basically there in inner floral organs and regulating it.To identify such kind of gene, the differential display RT PCR was taken up and then one ofsuch genes was identified which was OsMGH3.And it was shown that actually OsMGH3 was expressed in the floral, inner floral organs.So, this is again if you look, this is northern blot and if you look the expression patternof OsMGH3, you can see that in wild type, leaf sheath it is not expressed whereas, inthe young panicle, panicle is inflorescence in case of rice, it has a very high levelof expression.But if you look a panicle which is mutant panicle, where MADS1 is down regulated, theexpression of OsMGH3 also disappears.Which means that first thing that, there is a organ specific or tissue specific regulationof this gene.The gene is only expressed in the panicle, not in the leaf sheath and it is expressionor it is activation is dependent on the MADS1.So, this is one example of differential display RT PCR.Then subtractive hybridization was used in this study.Where they have done a comparative transcript profiling of gene expression between seedlessvariety and it’s seedy wild type during floral organ and development and I will notgo in detail.Then the technique which, I was going to talk about is microarray.So, as I said that microarray is a kind of chip.So, this kind of chip was generated and this was very widely used in the post genomic era,before RNA sequencing.Once the genome was sequenced, we identified the genes, we identified the sequence of allthe genes.Then those genes, the probes for those genes were designed and these probes were spottedin these chips.So, for every gene we had a kind of probe.And then there was two way of identify the differential expression, one was the two colorway and one color way.So, in one color way what you have to do, let us assume that, you want to test a wildtype sample which is your test or mutant sample.So, you want to identify the genes which are differentially expressed between this wildtype and this mutant.And what you can do, you can extract total RNA from the wild type, total RNA from themutants then you label this RNA.And one way of labeling was, the biotin labeled cRNA synthesis, both were basically labeled.So, entire RNA population in these two conditions, were labeled and then this labeled cRNA wereused to do the hybridization in the chips.So now, these chips contains, global genes all the genes represented in an equal amount.Now since let us assume that, there is a gene A, which is more in case of wild type, butit is getting down regulated in case of mutant.Then you are expecting more cRNA in wild type, less cRNA in mutant.And when you hybridize on the chip, you are expecting more signal for that probe in caseof wild type whereas, less signal in case of mutant.And then if you compare the signal intensity, you do the data acquisition scanning and ifyou calculate the absolute value of hybridization, you can identify the difference in the expressionpattern of gene A between wild type as well as mutant.And then, another way of analysis was that let us assume, the similar case you have wildtype RNA, you have mutant RNA and then you take total RNA from both the sample.And then you label them with two different dye for example, this is cy5, this is cy3,cy5 will give red color, cy3 will give green color and then you take equal amount of totalcRNA and hybridize on the same chip.So, basically the difference between these two methods was, here both the cRNAs, werecRNAs were labeled with the same label, whereas they are labeled with the different labels.Here they were probed, they were hybridized on two different independent chips, here bothare being hybridized on the same chip.So, let us assume if there is a chip which is for gene A. Now normally if the expressionlevel of A is same in wild type and mutant, then you are expecting same amount of cy 5label cRNA and cy 3 level cRNA to go and hybridize with this particular probe.So, you are expecting same amount of red signal, same amount of green signal and the resultwill be yellow signal here.But if any one of them are differentially present for example, if expression is morein the wild type then you are expecting more red signal than the green and then the colorof these spots will change, it will be more towards the red.If gene is down regulated then it will be more towards the green.So, based on this color and then the quantification you can actually calculate the fold changebetween wild type and mutant for all these gene.These techniques you can also use for the different stages, it is not only in wild typeand mutant.For example if you take this this particular case.So, where what people have studied?People have studied the genes which are differentially expressed, at different developmental stageof rice panicle development or flower development.So, for example, if you take these stages, this is stage 0 which is basically vegetativephase and this is your shoot apical meristem.Extract total RNA from vegetative phase and then you this is a transition from vegetativeto the reproductive phase.Then after transition, this inflorescence is undergoing a different stage of development.Here primary branch meristem and then secondary branch meristem, secondary spikelet meristemall these developmental stages it is going across.So, what people have done, they have taken RNA from here from all these different stagesand they have performed the microarray analysis in great detail.And this how they could actually identify several things, first thing they could identifythe gene which is present in a particular developmental stage, but absent in the developmentaldifferent developmental stage.Or a particular genes, how it’s dynamic expression pattern is across the development.For example, some genes might be 0 here and then they are suddenly getting activated andthey are maintained across the development.Or they are activated in this developmental domain and then they are going down.So, this is a basically dynamic expression pattern you can analyze, using this technology.And this everything, we are doing at the global level and we can identify.For example if, the some of the genes, they has been tested here.So, you can check this is RT PCR analysis.If you see the gene LAX, the gene LAX is very low at 0 stage but across the stages 1 2 34 5 the expression level is increasing, whereas if you take this FZP which is FRIZZY PANICLE,this is the expression starts or activates only from the stage 4 onwards and continuein the stage 5.It is not present in the early stage, whereas this MADS3 it is getting activated in stage5.So, this is how that microarray was extremely useful, in studying this kind of genome wideor differential expression analysis.This is another example of a microarray which was used and here you can look here that,they have focused their study on the MADS box containing genes.Why I am focusing most on the MADS box containing gene?Because this was the class, which is well established to regulate a lot of developmentalprocesses, they are very very important with respect to the plant development.And here they have taken large number of tissues mature leaf, roots, 7 day old seedling andthen this panicle, different size of panicle.Different size of panicle or inflorescence signifies different developmental stages incase of this one.And then they have checked the expression pattern, through microarray analysis.And what they can identify, there are lot of genes.So, if you look the expression is here.So, if green is less expression, red is high expression.And then you can look that a particular gene, if you go across the different organs youcan see their expression.How the expression patterns are changing from one developmental stage to another developmentalstage or one tissue to another tissue.So this, this was important study.Nowadays it is RNA sequencing, this is next generation sequencing it’s very cheap andvery easily available.So, now, people are doing RNA sequencing and RNA sequencing has some advantages over themicroarray.First advantage is that, for microarray to generate the chips you have to have the sequenceinformation, which means that the genomes of the organism has to be sequence, then andonly then, you can define probes for the chip.But in RNA sequencing, the genome even the genome is not sequenced, it can be studiedthrough RNA sequencing.Second important thing, one very very important process in eukaryotic organism is alternativeRNA splicing.Alternative RNA splicing is like, if a gene has multiple introns.So, let us assume this is intron 1, intron 2, intron 3.So, there are a large number of gene regulation occurs at the level of splicing.So, alternative splicing essentially provides, an opportunity to generate more than one typeof protein from a single gene, just by alternatively splicing the different introns.For example in one messenger RNA, you can generate from here if you splice all the introns.Then you can generate a messenger RNA, where you do not splice intron 1 or you do not spliceintron 2 or you do not splice intron 3 or you splice individual, you can even skip theintrons, you can splice this exon along with the intron.So, this provides a large number of different transcripts from the same gene.And it is very difficult or it is very challenging to keep probes for all splice variant in thechip, in the microarray experiment.So, it was very challenging because if you do not know, how many splice variant existsfor a particular gene, it is very difficult.Because normally when you are designing the chip, you used to keep one or maybe two ormaybe three chips for a particular three oligos for a particular gene.But if there is some intron here, you will not have intron or splice variant a specificchips.But in case of RNA sequencing, basically it can capture all the splice variants.So, if you look the technique how RNA sequencing functions?So, basically what we do in the RNA sequencing, you extract total messenger RNA and then youcan specifically remove messenger RNA from the total RNA.How you can do that?You all know that messenger RNA they have poly A tail at the three prime end and thenyou can use oligo dT tagged with some kind of beads.And using this system, you can specifically remove messenger RNA from the total RNA population.And then you use this RNA and you can do the fractionation you can make a small fragmentof RNA and then use some random primers.Random primers are basically small six nucleotide long single nucleotide primer with differentsequences.So, it can randomly go and bind different places and then you can make first strandcDNA, synthesis second strand cDNA synthesis.So, basically you are generating a different, different small fragments cDNA and it is doublestandard cDNA.And then one important thing, what you can do, you can put the phosphate, normally theprimers they do not have phosphate at their end.So, then you can put provide phosphate because if you have to use this for further ligationprocess, if they do not have phosphate at their end, then they cannot make a phosphodiesterbond.So, you can do the end repair put the phosphate.And another thing what you can do, you can add flanking A, this you can do using polyA polymerase and then if you add this A tail, this will provide you to take this fragmentand add some adapter so, you can ligate adapter.This adapter can have a stretch of T. So, T and A, they will go and they will annealto each other and then you can perform a ligation reaction.So, essentially what you are generating, you are generating double stranded DNA fragmentswith adapter at both the end.And then you know the sequence of this adapter you can use this adapter sequence and youcan take and sequence this entire cDNA population.This is very widely used these days and this can be used to identify.So, if you take two different population of RNA, two different population of then youcan generate two different population of fragmented cDNA.And if you sequence them and compare them, you can identify differential expression levelof different RNA.So, again I will give some of the examples, where RNA sequencing was used to identifydifferential expression pattern.For example, if you take this.So, here different stage fruits were taken and total RNAs were extracted and RNA sequencingwas done.And you can clearly see different genes and their expression pattern.For example if you look here.So, if you look this gene, or any of this gene, this has a very low expression at thisstage, but if you come here by the end of this stage the expression level is reallygoing very high.So, this tells, that there are a differential expression of different type of genes.And here you can see that, this is heat map of RNA sequencing data which involves theshoot development.So, there they have taken different types of shoots here secondary shoots, primary shoots,then there is some, something called mother variant shoots.And they have extracted total RNA and they have performed RNA sequencing, across thesedifferent types of shoots and they have identified different tissue specific genes which arepresent here.This is another example, where comparative RNA sequencing analysis was done for transcriptomedynamics during petal development in in Rose.One of the Rosaceae member and where you can see that they have taken different stagesof the petals from the different developmental stage and they have performed the RNA sequencinganalysis, and they could identify a different genome expression pattern.And you can see, they can identify here all these different types of transcription factor.Depending on your studies, you can focus on transcription factor.So, you can identify all the transcription factors which are differentially expressedor anything any other kind of transcription factor.Another way if you take this kind of RNA sequencing in RNA sequencing you can combine with a differentother technique.So, it is not stand alone technique and then you can perform a more specified or more definedway to generate RNA sequencing data.For example, if you have some kind of developmental stages maybe I will draw here.So, for example, if you want to study a developmental stage at a particular, let us assume thatyou want to study root developmental stage and you know that root development occursthrough different stages of the development.The first stage in the lateral root, branching is that the specification of some, some cells.So, these are the mature cells, now they are taking stem cell property and then these cellsstart the cell division.They undergo the process of cell division and they generate some kind of primordia whichis called root primordia.And then this root, root primordia undergo the process of cell differentiation.It start root specific differentiation program and then finally, the primordia is emergingout.Now, if you want to really identify the genes, you can collect this tissue very specifically.There are lot of techniques available, one of the technique which is called laser capturemicrodissection.You can use and you can collect all these primordia and then extract total RNA fromthis primordia and then you can identify.Or you can take some wild type sample and the mutant sample, were a particular transcriptionfactor a particular gene is missing.And you want to identify what are the genes, which are, whose expressions are getting affected,when you mutate a particular gene.Then you can take total RNA, you can do tissue specific, total RNA extraction.And then perform RNA sequencing to identify the differential genes which are present inthese two particular condition or two particular mutants.So, this is is analyzing expression pattern, where we have tried to study today how toidentify differential expression pattern.Now for doing spatial and temporal expression pattern analysis, there are several method,which we will discuss more in the next class.But to just briefly introduce them, one way of doing is that to study RNA localization,which we are going to do by RNA-RNA in situ hybridization.Then you can do protein localization, you can use some specific antibody and localizeyou can study their specific localization in across the tissues.And then you can do a kind of promoter trapping, where you can use a kind of promoter lessreporter gene.And you can identify if a promoter or you can do transcriptional fusion, you can dotranslational fusion.So, maybe we will stop this class here.And in next class we will discuss in detail, how to study spatial and temporal expressionpattern and functional characterization of gene through reverse genetics based approaches.Thank you very much.