Defensins were foremost identified as antimicrobic peptides and subsequently several lines of grounds suggest that they can interact with host immune cells, thereby playing of import functions in both innate and adaptative immune responses against bacterial infection. They are classified into I± , I? and I? Defensins ( Shi, 2007 ) . This thesis reports the probe of the look of Defensin cistrons ; I± Defensins 1, 4, 5 and 6 and I? Defensin 1, 4 and 5 in monocytic cell line, THP-1, utilizing rearward RNA polymerase polymerase concatenation reaction ( RT-PCR ) .
First experiments were undertaken to clarify the effectivity of RNA isolation from THP-1 cells exposed to LPS for upto 24 hours. Defensins mRNA look by monocytes is thought to be increased after activation with lipopolysaccharide ( LPS ) in a dose and time-dependent mode ( Duits, et al. , 2002 ) . Therefore, the THP-1 cell lines were pre treated with LPS for different times, 6 hours and 24 hours, so as to compare the uttered defensins from these cells with other cells which were non treated with LPS.
4.1 RNA extraction
The RNA was extracted and purified from the THP-1 cell lines utilizing Trizol and a mixture of phenol-chloroform. The RNA outputs vary depending on the, the culturing of cells, thoroughness of treating the sample and overall handling of samples as non to degrade the RNA.
4.2 RNA concentration and pureness
The concentration and pureness of RNA output was assayed spectrophotometrically utilizing the GeneQuant ( Amersham Biosciences ) taking the advantage of the ability of nucleic acid to absorb ultraviolet visible radiation with optical density upper limit at 260 nanometers.
Calculation based on the optical density of 260 nanometers provides small information about the quality and pureness of the sample as the presence of the extra salt, polluting proteins or the carryover of any dissolver may impact the optical density significantly ( Farrell, 2007 ) . Therefore, measurings of A260/A280 ratio and A260/A230 ratio were used as they provide better appraisal on the pureness of the dilution.
A pure RNA sample has A260/A280 ratio of 2.0 A± 0.1 and A260/A230 ratio should be greater than 1.7 ( Farrell, 2007 ) . The A260/A280 ratio for untreated THP-1 cells in this experiment was 1.59, 1.595 for cells exposed to LPS for 6 hours and 1.64, for 24 hours. These consequences are all below the expected scope of A260/A280 ratio ( 1.9 – 2.1 ) which indicate that the RNA output may non be pure. Similarly, the A260/A230 ratio obtained were lower than1.7. This may be due to many grounds. The truth of A260 optical density as an index of concentration and A260/A280 as a pureness index have been found to be interfered by pH of the dilution and is most dependable when measuring are made between 7.5 and 8.5 ( Farrell, 2007 ) . In this experiment RNase-free H2O was used, which pH value was non measured, while it is recommended to utilize buffered solution as the H2O frequently has an acidic pH and can take down the A260/A280 ratio ( Ambion, 2009 ) . The TE buffer, Tris and EDTA, could be used as an option to the RNase-free H2O ( Ipsogen, 2008 ) .
Lower A260/A280 ratio, less than 1.9, normally indicates protein taint while insulating the RNA and low A260/A230 ratio is largely caused by improper drying of sample or carryover of trichloromethane which may non be removed decently even after rinsing with 70 % ethyl alcohol. Therefore, the lower values might be due to the uncomplete remotion of protein from the cell lysate or carryover of trichloromethane or phenol used during isolation of RNA. In add-on, proficient mistakes while pipetting, fixing and commixture of dilution might besides impact the readings. As spectrophotometric method can non know apart between RNA and DNA, samples have to foremost be resuspended with RNase-free H2O ( Scheppler, et al. , 2000 ) . Therefore, the consequence might besides be affected by improper intervention of sample. Fluorometric checks can be used, as they are more specific and sensitive for quantitation and they can know apart between RNA and DNA, nevertheless, they do non supply information about the extract pureness ( Kieleczawa, 2006 ) . Droplets of H2O and bubbles inside cuvettes may interfere with the consequence and any dust atoms cause light spread at 320 nanometers and can impact optical density at 260 nanometer ( Avison, 2006 ) .
By and large when the concentration of RNA in the sample is low so, there are more opportunities of acquiring mistakes in the measuring. Therefore when the reading is significantly low so the dilution should be repeated to obtain a higher value. Normally when A260 value is below 0.1 more sample should be added and when higher than 0.5 so it should be diluted ( Avison, 2006 ) . In this experiment A260 obtained was within this scope which suggests that the dilution was acceptable. As it is sometimes hard and impractical to quantitate RNA isolated from little samples that will hold lower concentrations after dilution, other methods can be used. For illustration, “ fluorescent dyes such as RiboGreenA® exhibit a big fluorescence sweetening when edge to nucleic acids and every bit small as 1 ng/ml of RNA can be detected and quantitated utilizing RiboGreen with a standard fluorometer, fluorescence microplate reader, or filter fluorometer ” ( Rapley, 2008 ) . This method is comparatively insensitive to non-nucleic acid contaminations normally found in nucleic acerb readyings and so the consequences are normally non subjected to contaminations. However this method has restrictions as the RiboGreen reagent can adsorb to the sides of plastic tubings and has to be stored in the dark ( Ambion, 2009 ) .
4.3 RNA unity
The purified sample of RNA was checked for unity and quality utilizing 1 % agarose gel cataphoresis. Although the RNA concentration outputs were low, they were still sufficient for agarose gel analysis which showed the two ribosomal RNA ( rRNA ) bands, 28S and 18S, clearly as labeled ( Figure 6 ) . The 28S rRNA set should be about twice every bit intense as the 18S rRNA set. This 2:1 ratio ( 28S:18S ) is a good indicant that the RNA is wholly integral ( Figure 12A ) ( Rapley, 2008 ) . Partially degraded RNA exhibit a smeared visual aspect, with a deficiency of crisp rRNA sets, or will non exhibit the 2:1 ratio of high quality RNA and wholly degraded RNA will look as a really low molecular weight vilification. Different grade of smearing can be ( Figure 12B ) bespeaking RNA debasement. The gel cataphoresis consequence obtained from this experiment ( Figure 6 ) shows no smearing and the 2:1 ratio ( 28S:18S ) was obtained which indicates that the RNA in the samples is integral.
In this same gel, some other sets appeared before the 28S set in the country where normally DNA sets are seen. These may be DNA bands or taint from other beginnings. The storage of sample may impact the quality of the sample, nevertheless, this can be excluded as in this undertaking the extracted RNA samples were electrophoresed instantly after extraction and quantitation.
Figure 12B: Different grade of RNA debasement. Adapted from Nature, 2009.
Figure 12A: 28S and 18S from integral RNA. Adapted from Ambion, 2009.
Other methods can be used to look into RNA quality and unity, for illustration, Agilent Bioanalyzer and Biorad Experion, can measure the quality and integrates the measure of RNA in the samples merely and in a short clip. Hence, provides elaborate information about the status of RNA samples together with appraisal of RNA concentration and pureness. Less measure of sample is required during analysis of RNA and some have the installations to expose the consequence in a gel-like image and an electropherogram. However due to the disbursal of the hardware and consumables they are non used often ( Rapley, 2008 ) .
4.4 Synthesis of complementary DNA ( complementary DNA ) and PCR elaboration of ( complementary DNA )
The 3rd experiment was used to look into for mRNA look of defensin proteins utilizing RT-PCR through complementary DNA ( complementary DNA ) synthesis. The complementary DNA was used in RT-PCR reaction as templets for subsequent PCR elaboration utilizing specific alpha and beta Defensins primers. Controls were besides used to formalize the trial and aid to bespeak if any wrong processs had had taken topographic point ( Farrell, 2005 ) . No templet controls were included as negative controls which provide a mechanism to command for external taint or other factors that can ensue in a non-specific addition of the look. The most common housework cistrons used is Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) , used as positive marker in this survey, and I?-actin, has besides been used as a housework index which are ubiquitously expressed ( Nicot et al. , 2005 ) . The intent of analyzing look of the housework cistron is because it is thought to stay changeless, so normalising for fluctuations in processing and making of the look. When there is no signal from the positive control, so this indicates that RNA sample is hapless and unable to back up rearward written text or elaboration of sample ( Farrell, 2005 ) . PCR merchandises demoing the places of the different size categories of DNA fragment were clearly seeable under ultraviolet irradiation proposing that RT and RCR had taken topographic point.
4.4.1 Defensins look in untreated THP-1
As showed in figure 8, the marker worked good and gave a clear set at about ( ~ ) 250bp. The merchandise length, bands from untreated cells, were compared with the provided merchandise length of each primer. The untreated THP-1 cells showed no Defensins look except for I±-Defensin 1 which gave PCR merchandise length of about 110bp. The cells besides showed weak PCR DNA set with I±-Defensins 5 and the staying sets showed PCR merchandise less than 100bp. These sets, less than 100bp, are largely primer dimers, the merchandise of nonspecific tempering and primer elongation ( Abd-Elsalam, 2003 ) . Defensins were expected to be expressed by the THP-1 cells after the intervention with LPS as noted by experiments which reported that Defensins mRNA look by monocytes is increased after activation with lipopolysaccharide ( LPS ) ( Duits, 2002 ) . These consequences suggest that Defensins are non or ill expressed by THP-1 cells which have non been treated by LPS.
4.4.2 Defensins look in treated THP-1 cells for 6 hours
LPS treated THP-1 cells, for 6 hours shows PCR bands runing between 100bp and 200bp. By comparing these consequences with untreated cells consequences and the expected values ( table below ) it could be concluded that when THP-1 cells are treated with LPS it is implicative that they express Defensins messenger RNA. However, the look was non to the expected values except for the I±-Defensin 5 which gave value of ~190bp that is near to the expected value,179bp.
Expected length in base brace ( bp )
Obtained length in base brace ( bp ) , about ( ~ ) ,
for cells treated for 6hrs
Obtained length in base brace ( bp ) for cells treated for 24hrs
Table 4: The expected length of Defensins primers ( alpha and beta ) in base brace and the approximate obtained length for the cells treated for 6 hour and 24 hour.
The lower look from these cells might be due to deficient clip for the cells to turn or to absorb the LPS although this is extremely improbable. It might besides be that the 6 hours is non the ideal clip for THP-1 cells to show Defensins in sufficient sum likewise to the determination of United Nations expressed hBD2 by human tracheobronchial epithelial at 6 hour ( Becker, et al. , 2000 ) . In add-on, other factors including the quality of enzyme and RNA used for rearward written text and complementary DNA synthesis are of import issues in order to obtain high outputs of quality full length complementary DNA with full cistron representation ( Abd-Elsalam, 2003 ) . “ The optimum primer sequence and appropriate primer concentration are besides indispensable for maximum specificity and efficiency of PCR merchandise ” ( Abd-Elsalam, 2003 ) . Since that this experiment was conducted under ill optimized conditions including the primer used, reagent used, Magnesium degree and rearward RNA polymerase, hence, the consequence could be perchance affected.
It is besides clear that the DNA PCR sets are a spot smeared which might either be due to proficient mistakes or due to the complementary DNA merchandise that might be degraded or contaminated. Degraded RNA will non bring forth full-length complementary DNA and taint may suppress the synthesis of complementary DNA ( Farrell, 2005 ) . By and large when executing PCR elaboration, utmost attention should be exercised to forestall DNA taint. The GAPDH, positive control, gave a clear set and the negative control gave no set which are the expected consequences and could reason that the overall experiment was approached right.
4.4.3 Defensins look in treated THP-1 cells for 24 hours
Cells when treated with LPS for 24 hours show clearer grounds of look of the Defensins compared with cells treated for 6 hours. However, the look was still less than the expected values except for the I±-Defensin 5 which gave a higher than expected value, ~ 220bp, than at 6 hour, compared to 190bp. Interestingly the I±-Defensin 5 was expressed more than other Defensin types on the THP-1 cells either when treated for 6 or 24 hours. This suggests that when THP-1 cells are treated with LPS at 100 ng/ml for 6 and up to 24 hours they can show the I±-Defensin 5. These findings correlate with the survey of clip class for hBD2 in human tracheobronchial epithelial ( hTBE ) stimulated with LPS for different hours. The hBD messenger RNA was extremely expressed at 12 and 24 hours ( Becker, et al. , 2000 ) . It is besides clear that the cells expresses I?-Defensin 5 to a degree, 160, closer to the expected value, 172bp which suggests that it might needs more than 24 hours in order to be expressed as expected. Possibly if THP-1 cells were exposed to LPS for longer clip it might ensue in extra Defensins making the expected values of mRNA look.
4.5 The I±-Defensin 5 look in THP-1 cells treated with LPS for 24 hours
A farther measure was done in order to look into the look of I±-Defensin 5 individually. The PCR merchandise was run in triplicate from each type of THP-1 cells on 1 % agrose gel. Looking at the consequences from the 3rd experiment consequence, it was expected to acquire sets which indicate the addition in look as the cells exposed to LPS for longer clip. However, the consequence obtained from the gel showed no important difference in the look between untreated cells and those treated for 6 and 24 hours ( Figure 10 ) . This could be due to hapless quality of PCR templets, primers, reagents or any proficient mistakes.
The densitometry analysis of the PCR bands from the same gel was carried out in order to obtain statistical values which might assist in the probe of the affect of LPS in the look of I±-Defensin 5. The consequence ( Table 3 and Figure 11 ) showed low look of Defensin at 24 hours compared to the baseline and undistinguished addition after 6 hours of LPS which is opposite to the findings in 3rd experiment every bit good as other surveies that shows addition at 24 hours ( Becker, et al. , 2000 ) . This might be because PCR is inefficient or low sum of complementary DNA templet was used. It might besides be due to the consequence of ethedium bromide while staining the gel. It has been reported that gel concentration and gel thickness affect the rate of ethidium bromide staining since both retard the rate of diffusion of the discoloration ( Brown, 2002 ) . Furthermore, under-staining can greatly impact the densitometry of little DNA sets, below 500bp ( Ehrich, et al. , 2007 ) . Therefore, transporting out this experiment in optimum conditions including fresh sample and high quality selected primers could supply a better consequence in demoing the consequence of LPS in the look of I±-Defensin in THP-1 cells in different periods of clip.
The present survey is highly fresh because really small has been investigated into Defensins look in differentiated THP-1 cells. The noteworthy look is seen with alpha Defensin -5. The other Defensins species were expressed nevertheless, non to the expected value which could be related to many factors including the quality of sample, techniques, reagents non specific elaboration or dimers or even the cells themselves might non be ideal for showing these type of Defensins which could non be compared to other surveies as Defensins have yet to be reported for look in THP-1 cell lines.
The alpha Defensin- 5 which shows an addition in the sum of look after 6 and 24 hours could be the lone type of Defensin to be expressed by THP-1 cell lines. These consequences suggest that the look of the Defensins are non the same, even when same dosage and cells are used. This was besides noted by a survey which indicated that hBD-4 look was up-regulated by infection and in response to phorbol 12-myristate 13-acetate, but non in response to other inflammatory factors that up-regulate the look of hBD-2 or hBD-3 taking to reason that the initiation and antimicrobic spectrum are non same with different hBDs cistrons ( Garcia, et al. , 2001 ) . In another survey to look into the forms of Defensin mRNA look in malignant epithelial cell lines, A431 and M-Hela, by RT-PCR, showed that HBD-1 cistron is expressed merely in A431 cells, the look of HNP-1, HNP-3, HP-4 and hBD-2mRNA was absent in A431 and M-Hela while HD-6m RNA was expressed in both cell lines. It was besides indicated, on the same survey, that hBD-2 can be induced by challenge with cuticular growing factor ( EGF ) ( Lisovskiy, et al. , 2001 ) . These findings, hence, heighten the findings of this survey which suggest that cells merely show one type of Defensin species.
The look of Defensins may besides be induced by other constituents in add-on to LPS. A survey reported that viral infection can bring forth human I?-defensin-2 ( HBD2 ) and HBD3 from the epithelial cells ( Klotman, & A ; Chang, 2006 ) . Interestingly it is late found that their look is non merely restricted to microorganisms. In a survey to find the acute effects of drawn-out exercising on salivary degrees of defensin Human Neutrophil Peptide 1-3 ( HNP1-3 ) an addition in the concentrations and secernment rates of HNP1-3 following exercising was reported, nevertheless their Salivary antibacterial capacity was non changed ( Davison, et al. , 2009 ) .
Although the thesis reported that the alpha Defensin -5 seems to be the Defensin type expressed by THP-1 cells, farther verification trials have to be done in order to corroborate and analyze it including cloning and sequencing techniques or by Southern Blotting method.
The method used for look intoing the look of these cistrons was RT-PCR which can mensurate cistron look utilizing every bit few as one cell compared with Northern Blotting which requires excessively many cells to be used for cell sorting. It is besides provides dependable informations due to the efficiency of both RT and PCR ( Riedy, et al. , 1995 ) . Assorted research lab methods have been used to observe defensins expression utilizing method other methods than RT-PCR. For case, ProteinChipA® Array, surface enhanced laser desorption/ionization ( SELDI ) engineering combined with time-of-flight mass spectroscopy was used to observe I?-Defensins in biological fluid. This method offers several proficient advantages for sensing of Defensins in biological fluids, including easiness and velocity of showing, no demand for HPLC preliminary processing, and little sample size ( Diamond, et al. , 2001 ) .This article is non included in your organisation ‘s subscription. However, you may be able to entree this article under your organisation ‘s understanding with Elsevier. In add-on, Western Blot and Enzyme-Linked Immunosorbent Assay ( ELISA ) can besides be used to observe human defensins protein look ( Schneider, et al. , 2006 ) . Human beta Defensin 2 can be detected in patient ‘s stool samples utilizing I?-Defensin 2 ELISA kit and Immunohistochemical trials as it is found that I? -Defensin-2 lack can be observed in the enteric mucous of persons with Crohn ‘s disease ( Langhorst, et al. , 2009 ) . In add-on micro arrays, and Consecutive Analysis of Gene Expression ( SAGE ) have been besides used to look into cistron look.
As Defensins are known to supply broad scope of protection, it is hence, deserving to include surveies done to look into their stimulation in different cells and stimulators and correlate them with diseases. For case, surveies were done to analyse the molecular links between infection and the look of human I? -Defensin ( hBD ) -1, hBD-2, and hBD-3 in the human cuticle. The survey concluded that the three microbe-derived molecules LPS, peptidoglycan ( PGN ) , and streptococcic pyrogenic exotoxin B ( SpeB ) were all capable of bring oning or increasing the look of hBD-1, 2 and 3 in keratinocytes of whole human tegument ( Sorensen, 2005 ) .
The experimental attack could be enhanced by sing a assortment of other concerns.
4.6 Need for farther work
In this survey the concentration of the RNA was low, therefore it might hold been better to hold used other methods of obtaining RNA and to corroborate it ‘s pureness as the PCR technique is prone to taints. Another concern would be to seek to optimise the PCR reaction as optimisation can minimise usage of sample and reagents and maximise duplicability and dependability. The experiment may besides be done in different timing i.e. other than 6 and 24 hours which might non reflect alterations of cistron look either between these two clip points or before or after the designated clip points.