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Defensins are small naturally occurring antimicrobial peptides and therefore part of the AMP group. Research have shown they play an important role in fighting against predominantly / mainly bacterial infections in conjunction with / whilst working in sync with the host immune system and in turn involved in both innate and adaptive immune processes. It is said that there are 3 (naturally occurring) defensins groups, known as alpha (Î±), beta (Î²) and theta (Î¸). More research has gone into finding what the functions / how the defensins work and what its structure is / how it structurally looks of alpha (Î±) and beta (Î²) grouped.
The aim of this dissertation as stated before was to measure the gene expression of alpha (Î±)-1, 4, 5, 6 and beta (Î²)-1, 4, 5 defensins whilst being induced by a type of bacterial LPS (which is unknown) exposed for 6 hours and 24 hours (experimental tests) and no LPS (control tests) stimulation in human monocytic cell line of monocytic macrophage 6 (MonoMac 6 / MM6) cells and this being measured by using the technique / method of Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR).
The hypothesis of this study is that due to the type of cells being used in the experiment which are the human monocytic cell line MM6. There should be gene expression for alpha defensins irrespect of LPS stimulation or no stimulation. But with stimulation of LPS the gene expression of alpha (Î±)-1 defensins should increase in the MM6 cells when compared to the cells with no stimulation of LPS. In addition to that no type of beta (Î²) defensins gene expression should be viewed / visualised / obtained. (CHECK JOURNALS FOR MONOCYTES & BETA DEFENSIN GENE EXPRESSION).
In order to measure gene expression MM6 cells' RNA needed to be removed / extracted (from control samples - no LPS, experimental samples - 6hr exposured LPS and 24hr exposured LPS) effectively and efficiently as only with the good quality RNA can the study be taken further to making cDNA libraries stage. So therefore after RNA extraction it is important that the RNA purity and integrity be checked in order to move forward to the conversion of cDNAs from mRNAs. This was done / carried out by spectrophotometrically measuring the absorbance of RNA. Then getting the purity ratios required for A260 / A280nm and A260 / A230nm and compared against the normal reference ranges. Then the A260nm absorbances were used to calculate the total RNA yield from which certain amounts of RNA samples were allocated for the integrity testing by agarose gel electrophoresis. When the RNA showed that it was pure and integral in composition then the RNA was converted to cDNA and reverse transcribed by enzyme reverse transcriptase. Finally the cDNA was exponentially amplified by using the PCR method so that PCR products were formed which were then visualised by carrying out another agarose gel electrophoresis.
Therefore the results obtained throughout the experimental procedures are in Results Section and these include spectrophotometer absorbances of 260nm, RNA results of A260 / A280nm and A260 / A230nm ratios, the total yield obtained for RNA from calculating from absorbance results, gel electrophoresis picture to show the integrity of the RNA, PCR product gel electrophoresis pictures of MM6 (No LPS - Control, 6hr LPS and 24hr LPS - experimental) cells, densitometer results of bands present on the respective gel electrophoresis pictures and finally their densitometer bar chart analysis to show the gene expressions.
Interpretation of Data
First results obtained were spectrophotometer absorbance results of the extracted RNA from the 3 types of MM6 cells. From many years it has been found that the purity and calculating the total RNA yield obtained by carrying out spectrophotometer analysis due to the fact nucleic acids (RNA and DNA) absorb Ultra-violet (UV) light at the maximum wavelength of 260nm. In the experiment GeneQuant machine was used for the spectrophotometer analysis. The absorbance results obtained at 260nm for the respective RNA extracted from the cells doesn't give us any information as to how pure and the quality of the extracted RNA is as stated before that all RNA absorb UV light at maximum wavelength of 260nm. So by ----- (Person who found the ratios look in journals) who worked out that measuring absorbances at 260nm, 280nm and 230nmin turn changes into / have 2 types of ratios A260 / A280 and A260 / A230 that can give better indication as to how pure the samples are when comparing to reference ranges. These 2 ratio results also gives an idea if RNA samples have been contaminated by too much salt and protein concentration and solvent carryover which in turn affects / interferes in the maximum wavelength, the pure RNA absorbs.
So the reference ranges state that the ratios of pure RNA for A260 / A280 should be in the range of 1.9-2.1 (± 0.1) and for A260 / A230 should be greater than 1.7.
From Book by P. Jones - The use of spectrophotometer can also provide a check upon the purity of the RNA preparation. Measurement at 3 separate wavelengths, 230, 260 and 280nm, determines the ratio of polysaccharide: RNA: protein in a sample. Based on the average composition of RNA, the optimal ratio of optical density at 230, 260 and 280nm is 1:2:1.
While performing RT-PCR, preparation of quality mRNA is an important factor to be considered in order to obtaining meaningful gene expression (Fajardy et al., 2009). The quality of data analysis obtained from RT-PCR is strongly related to the integrity and stability of mRNA extraction (Fleige and Pfaffl, 2006). Thus, initial extraction of a good quality and quantity mRNA is essential and that, a great care must be taken while extracting mRNA in order to obtain a good quality product (Muyal et al., 2009). This is because unlike DNA, RNA is very labile bio-molecule and is much more susceptible to degradation by divalent cations, heat, elevations in pH and improper storage of tissue or cells prior to RNA extractions (Catts et al., 2005). Moreover, RNA can be easily denatured by the enzyme RNase presents on our hands and skin. As such, it is important to ensure that all equipments and materials are free of RNase at each and every step of the process and that the amount and quality of RNA is checked after extraction to ensure the high quality mRNA has been obtained (Fajardy et al., 2009). In this practical, a micro-capillary chip's electrophoresis was used to determine the quality and quantity of extracted mRNA. It is suggested that the mRNA purity ratio of great than 1.8 to be adequate (Muyal et al., 2009).
RNA extraction is a key and initial step in rt-PCR because the quantity and quality of the RNA that is extracted affects the rt-PCR results. RNA to be extracted from clinical samples generally are disparate and include blood and other bodily fluids (Bustin and Mueller, 2005). RNA is labile and isolation must be carefully performed which would ensure the purity and integrity and the removal of contaminating genomic DNA and PCR inhibitors (Valasek and Repa, 2005). RNA is susceptible to RNases that are present in all living cells and in order to obtain high quality RNA endogenous or exogenous RNases must not be introduced during the extraction procedure (Lee et al., 1997).
There are several key factors that determine the reproducibility and biological relevance of real time PCR results which include sample acquisition and RNA purification (Bustin and Nolan, 2004). Quality RNA is another key factor when performing rt-PCR. It is essential to minimise RNA degradation to obtain RNA free from contamination by DNA and proteins (Nolan et al., 2006). RNA purity also affects results obtained by rt-PCR. To analyse RNA purity in this study a micro capillary chip electrophoresis was utilized and the samples obtained RNA quality between 1.79 and 1.97 which was suitable to be assayed as the optimal ratio for RNA quality is approximately 2 (Sudgen and de Winter, 2008).
So the results obtained by MM6 cells are as followed: for MM6 LPS untreated cells the ratio for A260 / A280 was 1.851, for MM6 LPS 6hr exposed cells the ratio was 1.878 and finally for MM6 LPS 24hr exposed cells the ratio was 1.899. These results were slightly below the reference ranges for A260 / A280 of 1.9-2.1 which suggests the total extracted RNA is not of the purest but at the same time it can't be said that the RNA is fully contaminated as the ratio results are not far off the reference ranges. As for the A260 / A230 ratio, all are above 1.7 as respectively they had 4.397, 4.516 and 5.04. As mentioned before not just proteins, salts and solvent carryover interfere in the absorbance readings of RNA but pH as well. So the best range at which pH should be around 7.5-8.5 in order to not affect the wavelength of 260nm which is required when calculating the total RNA concentration in the MM6 cells and the ratio A260 / A280 which is used to measure the protein contamination and therefore important in determining how pure the RNA sample is. Throughout the experiment RNase free water had been used and generally it is said that water has an acidic pH. In this situation pH was not measured so for the slightly low readings this could have been one of the interfering factors. To get around this problem it is said to use buffers such as TE buffer, Tris and EDTA. But it should also be noted the buffer solutions interferes in the calculation of RNA yield and not the purity test. Therefore it would be best to do 2 separate tests, one for quantifying RNA and the other to check the purity of the RNA sample (Avison - measuring gene expression book).
Also Check Ambion website and Ipsogen.
As mentioned before high concentration of proteins and salts and solvent carryover can interfere with absorbance readings and this could also be the cause / be one of the reasons for the low A260 / A280 ratio. These contaminations could have occurred at any of the stages when RNA was being extracted from mm6 cells during the practical sessions. Problems could have occurred when chloroform or phenol and as much of the 70% ethanol were being removed from the homogenates and this could have resulted in carryover of the chloroform and then not properly allowing the samples to air dry after removal of 70% ethanol. Also after the chloroform was added, the homogenate had separated into 3 phases of organic, non organic and protein plug. It was important when the solvent phase was being transferred to a new eppendorf some of the protein plug got transferred along resulting in increased protein concentration again affecting the absorbance readings. These solvents have aromatic rings which interfere in the absorbance of the 260nm wavelength by RNA. Other problems could have been because of human errors (technical problems) such as pippetting, preparing and mixing of solutions which again affect the readings. Also mentioned before DNA absorbs at 260nm wavelength same as RNA and therefore the spectrophotometer machine would be easily be able to discriminate the absorbance value is due to DNA or RNA in a sample / between the absorbance values of DNA and RNA. Also any DNA contamination is thought to mainly affect the PCR stage when all cDNA is amplified including any DNA contaminants. It is thought that when DNA contaminations are present it is best to use DNase I enzyme to remove the DNA but then chloroform and 70% ethanol is added again and also have to re-air dry (Avison). But this becomes a long process. But it is also thought that suspending RNA samples in RNase free water helps (Kieleczawa). Another factor that can interfere with absorbance readings are bubbles formed while pippetting solutions in the cuvvettes and presence of dust particles as this causes the UV light to scatter around 320nm and therefore affects the absorbance of wavelength of 260nm (Avison).
Because of all these interferances of DNA, protein, salt contamination, solvent carryover, pH and technical errors they can be avoided by using commercial RNA isolation kits as they are more sensitive and reliable as long as the instructions are exactly followed. The only problem with the kits is they are very expensive (Avison). - May be give examples of good kits
Also there are 4 main types of RNA isolation methods used, 1) phenol-chloroform-isoamyl alcohol precipitation, 2) Lithium Chloride precipitation, 3) Column extractions, 4) patent methods in commercial kits - look at their +ve and -ve points.
The yield (for 10 million cells) of calculated total extracted RNA in no LPS sample is 957 Î¼g / ml, in 6hr LPS sample is 910 Î¼g / ml, in 24hr LPS sample is 814 Î¼g / ml. Compared to the yield in literature not sure - need to look up - remember some website.
Generally if the extracted RNA from the cells and their yields are low it is expected that the error rates in measurements are thus going to increase. So this is why it is stated / said when the absorbance for 260nm is less than 0.1 more RNA sample should be added but if the absorbance reading is greater / higher than 0.5 then the samples should be diluted (Avison). The absorbance results obtained were slightly higher than 0.5 but due to the small quantity of RNA extracted from the MM6 cells, it was deemed not suitable / possible to dilute the samples. And even if these results were repeated and absorbance values then appeared to be less then 0.1 more sample would be needed. This is why quantitating extracted RNA by the method of spectrophotometry is not good as the method is not sensitive and accurate enough and therefore there are other techniques available that can be used such as using fluorescent dyes like Ribogreen that can bind to less than 1 ng / ml of RNA sample and still form a fluorescent end product colour that can be measured / detected by using a microtitre plate reader (Rapley). The benefits of this method / technique is that the small RNA sample can still be quantitated accurately and results can not be affected by the non-nucleic acid contaminants such as protein, salts and solvent carryover. There are 2 main limitations to this method, one is that it is too expensive to use and the Ribogreen can absorb the sides of the plastic tubes and therefore effects the end results and thus needs to be stored in the dark (Ambion and Rapley)
Look this info up in Rapley and Ambion website
Other methods that could also be used to calculate / quantify the RNA yield are chemiiluminescence and autoradiograph. See RNA book by P. Jones
The purity (discussed in above section) and integrity of extracted RNA in determining its effectiveness is especially required in specialist methods / techniques such as northern blotting and cDNA synthesis where if any of the extraction of RNA stages not carried out properly would result in contaminations in DNA, proteins, salts and solvents and therefore can fundamentally have problems / effects the end results. This especially would be a big problem if the starting point the RNA sample already received is less in quantity or from a resource that can't be obtained easily again. This is why it is crucial that RNA extraction from cells be successfully carried out.
In mammalian / human cells there are three types of RNA species found where ribosomal RNA (rRNA) is the abundant species and therefore makes roughly 85% or more than 80% of the total RNA present in the source / sample or in this case the monocytic cell lines. This is the only RNA that forms discrete bands on the electrophoresis gel pictures. In eukaryotic cells there are 4 types of rRNA 5s (approx 120 nucleotides), 5.8s (approx 160 nucleotides), 28s forms part of the large 60s ribosomal subunit (approx 4700 nucleotides) and lastly 18s forming part of the 40s small ribosomal subunit (approx 1700 nucleotides) (Avison). The rRNA is made up of 28s, 18s and 5s. The transfer RNA (tRNA) and small nuclear RNAs (snRNA) making up 10% of the total RNA. The transfer RNA (tRNA) and small nuclear RNAs (snRNA) account for / form about the last 10% of the total cellular RNA. The last 1-5% is due to the mRNA which (RNA isolation by rapley) is especially important for this dissertation as using this form of RNA, can the defensins gene expression be measured. The coding RNA comprises / forms around 5% of all RNA and this is known as the messenger RNA.
The mRNA part of the total RNA in the cell is what is required and this can only be isolated / purified in a single method by a poly T column or after all the RNA is extracted from a cell it is then converted to cDNA by the addition of a poly A tail.
Therefore after the extraction processes of RNA in the gel electrophoresis stage all the RNA will be present and the predominate RNA species that will be visualised on the gel are only rRNA 28s and 18s.
Therefore after the extraction processes of RNA, gel electrophoresis has been used to separate the RNA molecules by their sizes and to assess the RNA prepared has not been degraded by RNases or contaminations with DNA or sometimes types of RNA storage results in smearing effects before carrying out the cDNA stage where any problems in the quality and quantity can have a detrimental effect on the end result. Therefore the integrity of the RNA is checked using the 1% agarose gel electrophoresis method.
Even with the low RNA yield calculated there was enough RNA required to carry out an agarose gel electrophoresis (pic 1 in section 3 - fig 3.1). The gel picture distinctly shows 2 main bands corresponding to the large 28s subunit and the small 18s subunit of ribosomal RNA even though there is clear evidence of pippetting problems in the wells (when transfer RNA sample it was harshly done) resulting in the not so clear and sharp (crisp) bands as would be expected from a pure RNA sample.
It is expected that the intensity of the 28s bands should be twice the size of the 18s bands i.e. has the ratio of 2:1 (28s:18s) and therefore indicating that there has not been any degradation of the RNA and that it is still intact. But in the gel picture (fig. 3.1) this is not the case and that still doesn't mean there has been degradation of the RNA as that would result in smearing of the bands, no sharp shape of the bands and no 2:1 ratio of the 28s:18s bands being followed. The only main reason why the bands have resulted in the way they are due to the pippetting when RNA was transferred into the wells of the agarose gel, a hole was made in the wells by the tip of the pippetter and this allowed a lot of RNA sample to leak out into the TBE buffer resulting in the not so intense RNA bands.
Give gel pictures to show the degree of degradation of RNA and compare against the high quality intact RNA bands.
Also it should be noted due to the fact mRNA only makes around 5% of the total RNA it has a lower quantity and that's why it does not appear or visually observed on the gel but if it were to be present on the gel it would be between the 28s bands and 18s bands. Also the tRNA that makes up 10% can be found on some gels but generally it is noticed that as they have smaller molecular weights they are found near the bottom and if the electrical current is not switched off in time the tRNA comes off the gel and therefore would not be visualised. In fig 3.1 it does seem to suggest the bottom light bands are of the tRNA. And finally if it is found that RNA samples have been contaminated with DNA, their bands appear at the top of the gel close to where the well indentation marks are found.
Another method that "can determine the total RNA concentration", detect DNA contaminations and other errors and finally measure the RNA integrity (RIN) value is the Agilent technologies bioanalyzer system. This system can give a detailed analysis of RNA, DNA and protein samples. Samples are loaded onto a plastic chip that has 16 wells similar to that of a micro-titre plate. This method uses the similar principles of electrophoresis separation but in a slightly different way. The wells are connected to their own anodes and are filled with fluorescent dyes that act as the staining solutions in electrophoresis. Each time a cathode is inserted into the samples in the well and separates them according to their molecular weight and with an electrophoretogram connected that measures the intensity of the lights emitted by the fluorophore that are on the dyes. The end results are in the forms of discrete sharp pointed peaks and when degradation occurs then broad peaks appear. This method is very beneficial as it provides detailed analysis of the sizes and the intensity of the samples rapidly. At the same time the machine requires a very minute / small amount of sample for analysis so beneficial for those important RNA samples that can't be obtained again / difficultly - thus little sample is loss. The only problem with the bioanalyzer system are that it is very expensive and only beneficial if running large numbers of samples (value for money) and are highly beneficial for RNA integrity sensitive applications such as micro-arrays and real time RT-PCR (Avison).
Show bioanalyzer graph here (from avison).
Can also list the other methods such as those mentioned in the RNA isolation book by P. Jones like the ultra centrifugation / sedimentation coefficient, sucrose gradient / isokinetic gradient and column eluting effects.
cDNA Synthesis and PCR amplification
After results for RNA purity and integrity show that they are good quality enough to be used in / carry cDNA synthesis stage. This part of the experiment is also important as now it will be known from the PCR product analysis on the gel electrophoresis of the mRNA extracted from the MM6 cells express the alpha (Î±), beta (Î²) defensins genes that encode for the defensin peptides.
As stated before the studies aim is to investigate the gene expression of defensins when stimulated by bacterial LPS and therefore a good method used to do so is Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) which is a powerful and efficient way in studying and analysing gene expressions even if it only gives semi-quantitative information at the expression level. Still it should be noted the method is very sensitive, versatile and provides results rapidly / quickly even when limited amount of RNA is available. Therefore this method requires accurate quality and quantity of mRNA so that it can be converted into complementary DNA (cDNA) with the use of a RNA virus in this case Moloney Murine Leukemia Virus (M-MLV) (Michael McPherson & Simon Moller - PCR book). The next stage is to amplify the cDNA which is used as a template for specific alpha (Î±) and beta (Î²) defensins primers that can bind to their specific genes on the cDNA. It should also be noted when designing primers they should be very specific and show 100% homology to the gene sequence and the forward and reverse primers should be free of any single nucleotide polymorphism in order to appear on the gel electrophoresis as 1 band (Hughes et al., 2007 - Rashid's dissertation). Not sure to add this sentence as received primer dimmers on the gel picture - The defensins primer parameters match all the specified requirements.
The next most important thing is in order to validate the method being used, positive and negative controls must be used for many different reasons such as it helps to make sure that incorrect procedures have not occurred and therefore affecting the end results and to ensure that there is normalisation between samples i.e. the use of housekeeping genes such as Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) and Beta (Î²)-Actin and their gene expression should remain constant / consistent at all times irrespect of the changed environmental conditions / variables being investigated in this case bacterial LPS added to MM6 cells (Radonic et al., 2003 from Saeed's Dissertation). In this dissertation only GAPDH was used as a positive control and it should be noted that as stated before if the RNA extracted is not of good quality then the cells will not express the interested gene and other common / regulatory / housekeeping genes (Farrell 2005 - Munira - find journals for this).
So looking at the 3 PCR product gel pictures (fig. 3.2, 3.3 and 3.4) of MM6 (No LPS, 6hr LPS and 24hr LPS), all experiments have shown to work / have worked as this was confirmed by the presence of the bands of GAPDH and this also confirms the RNA extracted is of adequate state / quality as a common housekeeping gene expression worked. Also it was expected that GAPDH product molecular sizes / lengths should be around 200 base pair (bp) because of the designed GAPDH primers and that has occurred on all 3 gels. (Densitometer analysis of GAPDH were carried out and it has shown that their gene expression have been constant). All the negative controls have also worked as only primer samples (Forward and Reverse) and PCR reagent master mix were added so that's why see / visualise those faint bands. Therefore positive and negative controls have validated the experiments (to be satisfactory???).
Table for obtained PCR product lengths of positive controls, negative controls and alpha (Î±), beta (Î²) defensins genes. Either put here or in results section may be after the PCR gel pictures.
Defensins expression of MM6 cells not treated with LPS
Looking at the PCR product picture (Figure 3.2) and PCR product band lengths table (Section 3 or section 4) for MM6 cells not treated with LPS, two types of molecular markers were used and both clearly showed the sizes of the bands. Therefore it was easier in comparing the bands of the MM6 (no LPS) cells with the expected primer product lengths of all of the defensins genes. As mentioned before GAPDH band appeared around the 200bp mark which was expected. The negative control containing only the primers therefore that was below the 100bp mark. The only other clear band similar to the GAPDH was of the alpha (Î±)-1 defensins which was expected to appear at the 364bp mark but was found to be approximately around the 340bp. So this was not too far off the expected product length. All the other defensins had faint bands especially alpha (Î±)-5 defensins which appeared around the 150bp when its expected product length was 179bp and the same for beta (Î²)-1 defensins which appeared around the 300 / 310bp and was expected to be at 335bp. May be the problems with these bands could have been the PCR conditions and that's why they appeared faintly. Sometimes through trial and error it could be known what are the accurate / exact PCR optimisation conditions required for good PCR product bands to appear on the gels. With regards to the rest of the defensins bands they all appeared below the 100bp mark and appeared very faintly similar to the result of the negative control. It is thought the main reason for this is the primers have joined to itself and formed primer dimmers during the PCR annealing and elongation steps (Abd-Elsalam, 2003 and eBook on the laptop). It is thought the best way to avoid this is by using more cDNA sample and less forward and reverse primers. This would allow more chances for the complementary primers to bind to the sequence they were designed for and therefore allowing the possibility for a band to appear on the gel. A study by Duits suggests that when monocytic cells such as MM6 are exposed to LPS for a certain period of time this then causes the defensins genes to become activated and expressed and resulting in defensins peptides being produced to fight the foreign micro-organism. But this doesn't explain why alpha (Î±)-1, faint alpha (Î±)-5 and beta (Î²)-1 defensins appeared on the gel. (Need to look into this point - may be find more journals???)
Defensins expression of MM6 cells exposed to 6hr bacterial LPS
Again here the 2 molecular markers appeared clearly with some smearing making some of the bands appear faintly but still showed the different molecular weight ladders. The positive and negative controls have also shown to work. So when comparing the defensins bands against their expected values again only alpha (Î±)-1 defensins had a clear strong expression band appearing roughly around the 340bp which is not far off the 364bp actual length. Also when looking at the alpha (Î±)-1 defensins bands of no LPS MM6 and comparing against the 6hr LPS MM6, the band appears more stronger and denser in picture of 6hr LPS suggesting and confirming what Duits stated when once the cell is stimulated by LPS, the gene expression increases. So the densitometer analysis will be required to prove this point which will be discussed after looking at the 24hr LPS MM6 gel results.
Once again a faint band appears for alpha (Î±)-5 defensins around the 150bp mark. But all the other defensins have faint bands appearing just around 100bp and below 100bp. This again suggests that primer dimerisation has occurred. So both the forward and reverse primers need to be reduced in quantity for each defensins gene and in addition to that optimisation steps should be carried out in order to allow the other defensins bands to appear. It is also thought that another reason for the low molecular weights could be because of not allowing long enough exposure times of 6hr for the LPS to increase enough gene expression by stimulation and therefore not easily allowed to be visualised on the gel. A study by Becker, 2000 has shown that human tracheobronchial epithelial cells require more than 6hrs in order for the expression of human beta defensin 2 (hÎ²D2 to appear which did not at 6hrs. As stated before the quality and intensity of the RNA (mRNA) play an important part when reverse transcription stage is being carried out of conversion of mRNA into cDNA and therefore allowing the primers to bind to the full gene resulting in the band appearing on the gel (Abd-Elsalam, 2003). This is why optimal primer sequence, appropriate primer concentrations, correct magnesium levels and right amounts of other PCR reagents and Reverse Transcriptase enzyme needs to be used in order to maximise the efficiency and specificity of the end product and again this can only happen / occur when the trial and error of PCR optimisation occurs (Abd-Elsalam, 2003). The PCR bands that are slightly smeared could be due to degradation or contamination of the RNA during the extraction stages and this is also a factor thought to decrease the synthesis of good quality cDNA from RNA. But this could not be fully agreed / could not be the case here as the positive control (GAPDH) and alpha (Î±)-1 defensins worked so why did they not get affected by the RNA. Therefore overall the experiment has shown to work and the other defensins may need better conditions in order to allow the bands to appear as strongly.
Defensins expression of MM6 cells exposed to 24hr bacterial LPS
Lane 1 molecular marker bands are clearly visible but lane 2 molecular marker bands are starting to fade / be faint (near the bottom - lower molecular sized bands) and the bands slowly have parts slightly overlapping. With regards to the positive and negative controls they appear as similar to that in the other two gel pictures. Here again alpha (Î±)-1 defensins appear around the 350bp which is still close to the 364bp. Alpha (Î±)-5 defensins again appears faintly around 180 / 190bp which is either on spot its expected molecular size or slightly above the actual length. The rest of the defensins appear just around 100bp and below it. Again it could be due to the fact that the cells require a longer exposure time with LPS in order to increase the gene expression. As now you can see alpha (Î±)-5 defensins bands are slowly slowly becoming more visible from 6hr LPS to 24hr LPS therefore this could be due to the longer time the cell was exposed to LPS (more than the 6hr period). The study by Becker also states that the hÎ²D2 became expressed in the epithelial cells around 12 and 25 hours. Now looking back at the gel photo even beta (Î²)-5 defensins has a faint band is appearing around the 150bp mark which is also close to its actual product length of 172bp. So this also implies all the defensins except for alpha (Î±)-1 require more exposure time with LPS in order to appear as a proper band. Also again due to some of the defensins becoming primer dimmer it is necessary the PCR conditions optimised to help enhance the gene expression.
- Mention alpha (Î±)-1 densitometer analysis.
If time and finances were available / were not an issue the three cells would have had their RNA extracted again from newly cultured MM6 cells and maybe using a reasonably priced commercial RNA isolation kit to extract the RNA and maybe also using a micro-capillary chip's electrophoresis to measure their quality, integrity and quantity. And then tried to optimise the PCR conditions that could help the gene expression appear more visibly / visually. And if there were still similar problems to the current study, it would be suggestive that the cells require more LPS exposure time or these cells are / do not express these specific defensins genes or require other types of stimulants that can initiate the gene expression such as / fungal antigens or some of these defensins require the support of similar molecules like the toll like receptors to help induce the expression when cell is in contact with LPS or other types of foreign antigens.
Densitometer analysis were carried out on the GAPDH and alpha (Î±)-1 defensins genes as only these two produced strong clear PCR product bands. Additionally, in order to determine the effects of LPS on defensins gene expression relative to no LPS stimulation to the gene expression, statistical analysis needs / needed to be carried out in order to analyse the differences between the results which are visualised as significant (Avison). Ideally the experiment should have been carried out more than three times but due to the time constraints and resources problems that could not be possible. The gels of each type of cells were repeated twice and then using the densitometer to analyse the density of the bands and using these numerical results a bar chart was plotted. The bar chart showed the GAPDH generally had consistent gene expressions in all the cells (with and without LPS) which are expected as it is a common housekeeping gene. But with the alpha (Î±)-1 defensins gene, expression increased significantly by about 20% from untreated LPS to 6hr treated LPS MM6 cells. Then from 6hr treated LPS to 24hr treated LPS in the MM6 cells, the gene expression decreased by 5%. These results are different to the results obtained in the study by Becker which suggested that the beta (Î²) defensins gene expressions increased after 24 hours. This could have been due to many reasons such as effects of ethidium bromide on the gel after staining. Also the concentrations and thickness of a gel also affects the rate of diffusion of the ethidium bromide staining stage (Brown 2002). And in a study by Ehrich, 2007 suggests that under staining a gel affects the density of smaller bands that are below 500bp and therefore affects when / during / carrying out densitometer analysis. Also if insufficient PCR product was produced this is thought to be due to the low amounts of cDNA sample which in turn is thought to be the reason / cause of low / reduced gene expression. This is why optimal conditions are required when carrying out the experiment such as using good quality RNA for the cDNA conversion stage and good quality defensins primers that can show the effects of LPS has on the gene expression of defensins at different exposure times.
In this study it is viewed that only alpha (Î±)-1 defensins appeared clearly on the gels and was expressed with and without LPS stimulation. Faint bands of (Î±)-5 defensins, beta (Î²)-1 defensins and beta (Î²)-5 defensins were also viewed but may be due to the quality and integrity of the RNA, human errors in carrying out the technical work, primers may not have been specific enough to bind their target sequence or too much quantity of primers added to the sample resulting in non-specific amplification in turn resulting in primer dimmer formations, PCR method may have required optimisation and any other reason could be that these monocytic cells either require other types of antigens to stimulate the defensins expression or require support from molecules such as toll-like receptors (TLR) to induce / initiate a response. Also some of these defensins are said to be expressed in epithelial type cells as it is found generally a lot of beta defensins expressed by epithelial cells and a lot of alpha defensins expressed by paneth cells. There has been less research carried out on this specific type of monocytic cell lines MM6. So therefore it is difficult to compare the results found in this study to other studies of this sort.
Looking at the results obtained the defensins genes were not expressed similarly even though / when all the cells were of one type and had been stimulated with the same quantity of 100 ng / ml of bacterial LPS. These are similar findings to a study by Garcia stated that hÎ²D-4 expression when in response to phorbol 12-myristate 13-acetate was it then up-regulated by infection, but hÎ²D-2 and hÎ²D-3 were not up-regulated by any inflammatory factor suggesting that all hÎ²D genes are induced by different anti-microbes. In another study by Lisovskiy found that malignant epithelial cell line A431 expressed only hÎ²D-1 and HD-6 mRNA was found in both types of epithelial cells A431 and M-Hela. But hÎ²D-2 required to be induced by epidermal growth factor (EGF). And all HNP-1, 3 were not expressed in either of the malignant cell lines. Both these studies also agree with the statement that all the defensins genes are not similarly expressed in one type of cell and sometimes required to be induced by some kind of inflammatory factors or molecules.
Also a study by Klotman and Chang found that hÎ²D-2 and hÎ²D-3 were expressed in epithelial cells when they were infected by a virus. Also a study by Davison showed that defensins not only can be stimulated by infections from bacteria and virus but prolonged exercise which increased the expression of human neutrophil peptides 1-3 (HNP) and this did not have any affect on the bacteria in the salivary glands.
Even though the gel electrophoresis results found that alpha (Î±)-1 defensins was properly expressed and alpha (Î±)-5 defensins, beta (Î²)-1 defensins and beta (Î²)-5 defensins faintly expressed. This still does not mean the bands that appear are of alpha (Î±)-1 defensins was properly expressed and alpha (Î±)-5 defensins, beta (Î²)-1 defensins and beta (Î²)-5 defensins and so forth. This is because the primer could bind to other parts of the cDNA template and that's how the PCR product appeared. So in order to confirm that the bands are corresponding to the specific defensins genes they should be tested by using techniques / tested against techniques such as gene cloning and gene sequencing such as Southern blotting.
In this experiment in order to study the gene expression patterns of defensins when stimulated by LPS, RT-PCR method was used. This method generally is used to measure gene expression in one type of cells where as methods such as Northern blotting can use a range of cell types. As mentioned before RT-PCR is efficient, very sensitive, quite reliable and provides gene expression information rapidly (Avison / PCR book by McPherson). There are many different types of methods available / that can be used to measure gene expression. The specific methods sited in studies used to measure defensins genes are ProteinChip Array, Surface enhanced laser desorption / ionization (SELDI) technology and time-of-flight-mass spectrometry used to detect beta defensins in biological fluids. This technique is good at detecting beta defensins in fluids as it can rapidly / easily be carried out on small samples without having the need to use HPLC technologies (Diamond, 2005). In a study by Schneider the methods used were western blotting and enzyme-linked immunosorbent assay (ELISA). Also microarrays and serial analysis of gene expression (SAGE) have been used. In a study by Langhorst, beta (Î²) defensins 2 ELISA kits and immunohistochemical tests were used on patient stool samples and found that when a person has Crohn's disease Î²-defensins 2 is not found / viewed in intestinal mucous.
The functions of defensins are to protect the host cells against foreign pathogens and infection. On this basis many research has been carried out on different cells and different cell stimulants and then the results were compared to cells in diseased sates. A study by Sorensen tried to find a link between 3 beta defensins (1, 2 and 3) in human epidermis cells against 3 types of stimulants that can cause infection (microbe derived molecule LPS, peptidoglycan (PGN) and streptococcal pyrogenic exotoxin B (Spe B)). The study found that the 3 stimulants were able to induce an expression of hÎ²D-1, 2 & 3 in keratinocytes.
This experiment needs to be repeated again as the RNA obtained was not of best quality and purity and therefore contaminants from this stage affected the PCR stage and some of the product bands did not appear clearly. This is why the PCR method also needs to be optimised by changing the volumes / concentrations of the reagents to ensure that reliability and reproducibility of the results are increased. Also designing new sets of defensins primers to ensure that primer dimmers do not form again. From the different research studies it has shown that 6hr and 24hr may not be enough times for the monocytic cell to express defensins genes when exposed to the LPS for the stated times and therefore using other times like 12hr, 36 hr and 48 hrâ€¦etcâ€¦ to obtain meaningful results.
Need to do further work:
After making those experimental changes and hopefully this improves the results obtained / that has helped to improve the results obtained. These new results / they should be confirmed by sequencing techniques and gene cloning. And when the results are confirmed it is important to actually view the cells express genes and can they also produce functional defensins peptides so this should be carried out / done by using protein synthesis techniques and sequencing methods such as Western blotting.
This experiment concluded that alpha (Î±)-1 defensins were expressed in (all) MM6 cells when induced / stimulated by LPS for 6hrs and 24hrs. Other defensins genes such as alpha (Î±)-5, beta (Î²)-1 and 5 were faintly expressed. Therefore experimental changes need to be carried out to receive / obtain better results. The next stage of the experiment / study would be to confirm these and to carry out protein synthesis to confirm functioning defensins peptides are made by the cell. If sequencing results also confirm that the cell expresses alpha (Î±)-1 defensins gene then this would correlate / this correlates with literature which have found that in monocytes alpha (Î±)-1 defensins are expressed when induced by LPS, only thing would be that many studies have not used MM6 type of monocytic cells in order to find the expression of defensins genes.