Sample-2

5 Oct

Sample-2

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Analysis of gene expression by RNA extraction and Reverse Transcription Polymerase Chain Reaction

Role of Lipopolysaccharides (LPS) on cytokine expression in monocytic cells:

The bacteria’s haves LPS (Lipopolysaccharides) in their cell walls which induces an inflammatory response further leading to sepsis pathogenesis, inflammatory responses inside the body as well as the probability of autoimmune disorders. (Heinzelmann, M 2000).

The researches have not represented a clear picture about the mechanism involved in initialing the inflammatory responses but the reports have suggested that the monocytic cells are largely responsible for in the pathogenesis of LPS induced syndromes. LPS is the component of bacterial cell which is the key factor for regulating the biological responses by CD44 in monocytic cells during the immune and inflammatory responses.

The response generated by LPS initiates the responses in monocytic cells which release large quantities of pro-inflammatory cytokines and adhesion molecules like B7, ICAM-1 (Intercellular Adhesion Molecule 1), CD44(1-3) and VCAM-1( vascular cell adhesion molecule 1 ). (Verhasselt, V 1997)

The inflammatory responses are marked by the extra release of the pro-inflammatory cytokines, formation of adhesion molecules and co-stimulatory effect produced by them.

The alterations produced in the generation of CD44 expression is a key factor inj modulating the cell adhesion, inflammatory responses and the migration of molecules. (Levesque, M. C.,1997)

The Adhesion molecules are playing an important role in generating the inflammation responses by generating responses like mediating the leukocyte- endothelial cell adhesion, T cell- APC interactions and leukocyte migrations. (Pure, E 1999). The induction of responses produced due to CD44 expression is the major event responsible for migration of the cell to the sites of inflammation or injury. (Siegelman MH,1999).

The alternate mRNA splicing and posttranslational modifications are responsible for generating the extensive molecular heterogeneity which is the cause of the multiple functional attributes of CD44. (Dougherty, G. J 1994), (Katoh, S.,1995).

It has been researched that in monocytic cell lines as well as in primary human monocytes LPS is inducing the CD44 expression (. Levesque, M. C 1999).

Use of Tissue culture for measuring gene expression

The basic characteristic of complex tissues like the tumors, healing wounds and th stem cells is their functional heterogeneity.( Raaijmakers 2008), (Januszyk, M 2013). These different functional heterogeneities are because of the variations in both proteomics as well as cellular transcriptomics. (Maheshri, N. 2007).

Traditional methods used for the transcriptional analysis like the DNA microarray method etc majorly base its principle on collecting a pool of mRNA from a huge number of individual cells and therefore report the population on a wide average.

So far this technique has been widely successful at increasing our knowledge in understanding the physiologies of various diseases over the two decades but these methods lack the preciseness to observe the difference in expressions amongst cell of the target population. As a whole an expression can be read by observing the average response but each response cannot be singularly observed and singular response by cell subjects are therefore left ignored. (Eisen, M.B 1998).

The recent advances suggest that the limitations observed in the technique hamper the importance of the technology to observe and interrogate each cellular subgroup and therefore the dynamics concluded for the complex cellular subgroup is improperly concluded.( Levsky, J.M 2003).

In the recent years we have experienced the latest technologies like the Fluidigm Biomark system which has helped in interrogating each and every cell by resolution phenomenon. (Warren, L, 2006).

These evolved techniques have been successfully used in the laboratories nowerdays to find the solution about the fundamental principles underlying the mechanisms or physiologies of the tumor evolution, stem cell formation and evolution and pathologies of many chronic diseases and disorders. (Januszyk, M, 2014).( Suga, H.; 2014).

The basic question arises that the designing of such studies should be based on isolation of cells from the tissue by the primary culture or they can be subjected to the prior expansion of the complete cell culture.

This is an important step in case the population of the cells to be tested in very small or the insufficient quantity for testing makes it a difficult procedure whereas we have heavy cell lines also available in contrast to them.

The long term hematopoietic cell studies of the bone marrow which represents 0.0001% of the bone marrow can be easily studied by using the cell expansion techniques.(Wilson, A ,2008)

Use of Reverse Transcriptase and PCR controls:

Defining the positive and negative controls are an integral part of the lab tests. The usage of RT-PCR assays has been rapidly increasing since past years and the importance of such technologies has emerged as an important parameter in research and diagnosis studies. The risks of the false positive results created in PCR assay are due to the contamination in the amplified DNA products.

The methods are used to prevent the contamination and monitor the multiple negative controls. It is an odd fact that the false negative results often create a problem in the highly sensitive technique of RT-PCR which is identified at a lesser extent. (Macintyre E, 1997)

The researchers are more tempted to use the gene beta-actin fragments as the positive controls while performing the RT-PCR assays. (Reverse Transcriptase- Polymerase Chain Reactions). (Taylor JJ, 1994).

It is weird that the researchers are still relying on the housekeeping genes while using the RT-PCR technique despite of the claim which has been proven many times that the beta-actin gene might not serve as a valid RT-PCR control. (Soutar RL, 1997)

Role of GAPDH gene expression:

The control genes must be expressed in the cell-cycle in an independent pattern. It is very clear that the control genes must not be expressed at high levels when the gene is under investigation because the samples are eventually degraded partially. The amount of the specific target used could be below the PCR detection limits whereas the control gene may still show the positive amplification signals.

Such conclusions will suggest the presence of the limited quantities of amplifiable RNA/cDNA and therefore lead to false results or negative results. Some frequently used genes like Beta- actin and GAPDH are used as the control genes therefore they don’t provide useful controls in cases where the pseudogenes are also present. (Garbay B, 1996)

A good or high expression of the control genes can be created by amplifying the target genes on many PCR cycles. The researches have proved that using single –step amplification of ABL or BCR genes have provided good control in the diagnostic assays of PCR. (Hochhaus A 1996)

Assumptions of the experiment:

The application of polymerase chain reaction based techniques and methods involve some assumptions which are used so as to make an experimental design possible.

Using of appropriate controls for performing the PCR is also essential. The random amplified polymorphic DNA has been widely used by some researchers in RT-PCR to infer the phylogenies but the technique of PCR limits the use RAPDs. (Backeljau T 1995), (Smith JJ, 1994)

The mutation of the primer binding sites may result in the loss or gain of bands therefore the fragment size may be disturbed. The character homology may be violated by the RAPD polymorphism which will therefore not comply with the assumptions of the RT-PCR. (Smith JJ, 1994)

Accurate sizes (bp) of the PCR fragments:

The use of polymerase for PCR is basically application dependent. The majority of PCR amplifications use or require product size (amplicon) of 500 base pairs or less. Therefore for this product size negligible error rate of Taq Polymerase occurs and the mechanism works ideally. The historical as well as the conventional choice of reagent remains the same for Polymerase Chain Reaction which is Taq polymerase.

A mixture of Taq and other polymerases may be preferred if the long-PCR has to be performed where the amplicons are several kilobases in length. (Barnes WM 1994)

DNA sequence generated:

GAPDH F ACC ACA GTC CAT GCC ATC AC

GAPDH R TCC ACC ACC CTG TTG CTG TA

TNF α F CGA GTG ACA AGC CTG TAG CC

TNF α R GGT GTG GGT GAG GAG CAC AT

Housekeeping genes:

The housekeeping genes are identified by the technological models for any particular gene expression analysis using RT-PCR. The genes identified by the model are PGK1, GAPD, GUSB, TFRC, and 18S rRNA.

The housekeeping genes are basically used as the endogenous reference genes in the quantitative Real Time – PCR. Usually these housekeeping genes are expressed by all the cell types and the experimental conditions don’t vary them. (Tanja Pascale Neuvians 2005)

The paper focuses on the housekeeping genes which are used to express the Role of Lipopolysaccharides (LPS) on cytokine expression in monocytic cells. The favorable genes used are GAPDH (glyceraldehyde 3-phosphate dehydrogenase) or ACTB (beta-actin) as the reference genes for normalization in PCR. (Armin P Piehler 2010)

                   The other good combinations used for normalization in LPS- stimulated monocytes are PPIB (cyclophilin B), B2M (beta-2-microglobulin) and PPIA (cyclophilin A) which will be best expressed.

The Normfinder has suggested that TBP (TATA-box binding protein) and B2M are the best possible combination in this case.

When compared with these combinations the normalization method using the GAPDH alone has resulted in a significant higher change in the IL10 (interleukin 10) and TNF-α (tumor necrosis factor-alpha) expression. (Armin P Piehler 2010)

The gene GAPDH encodes a member of glyceraldehyde-3-phosphate dehydrogenase protein family. This encoded protein has been identified by the researchers as the moonlight protein (capable of performing multiple functions). The product of this gene is catalyzing the crucial energy generating step in carbohydrate metabolism.

In the presence of nicotinamide adenine dinucleotide (NAD) and inorganic phosphate. The product also functions in the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate.

In the nucleus this encoded protein has also been proved to have the uracil DNA glycosylase activity. (White MR 2015)

Design a set of primers:

GAPDH F ACC ACA GTC CAT GCC ATC AC

GAPDH R TCC ACC ACC CTG TTG CTG TA

TNF α F CGA GTG ACA AGC CTG TAG CC

TNF α R GGT GTG GGT GAG GAG CAC AT

Various methods for investigating gene expression:

The processes occurring inside the cell like cellular specifications, migrations, proliferations, apoptosis, formation of tumors, neurodegeneration and differentiation etc majorly involve the cell granules as an important route in governing and controlling these gene expressions. The better understanding of these processes also involves the diseases occurring inside the brain (cerebellum) and other parts of the body development and pathologies. (Savill RM 2005)

The gene expressions can be studied by using mouse as the model of research. A method used for appropriate results can be creating a conditioned mouse model where the loss of gene expression is totally restricted in the granule cell. The mouse model is little costly and time consuming but the results of the gene expressions are very appropriate and precise.

Another useful technique for gene expression is Organotypic slice cultures which are cheaper comparatively.  In this technique the tissue context is highly maintained throughout the experiment. The recent researches using the Organotypic slice cultures have gained great insights of the brain damage as well as brain development. (Rebecca M. Savill 2005)

The in-situ hybridization technique is an excellent technique for mRNA detection. In this method the cell type specific promoters are fused to the reporter genes therefore providing a spatial information at the cellular levels for further gene expression studies. While studying the gene expression and patterning in  Dictyestilium mutants the whole- mount in-situ hybridization technique was used. In this technique a blue colored enzymatic reaction occurs which forms a precipitate therefore concluding the reaction and the visualized precipitate can be studied. (Maeda, M., Sakamoto2003) (Shimada, N., Maeda, M (2004)

Reporter genes can be used as parameter to get an insight of the gene expressions. The technique used is to convert the reporter genes into clones in the plasmid vectors which is under the transcriptional control of various gene promoters. Now constructs are formed in the cell where the expressions of the reported genes are easily read and detected. The expression pattern observed by the gene indicates the activity of the reporter gene inside the cell. (Wang, N 1996) (Thompson, C. R. L 2004)

The immunohistochemistry method can also be used a s a technique for studying the gene expressions where the cells are stained with dyes which are then tagged by the suitable antibodies. The visual change in responses by the genes is easily observed and can be detected using the immune histology techniques. (Jermyn, K. A., Duffy, K. T.(1989)

Display the results that were obtained. (approx. 450 words)

The PCR was run for studying the effects of LPS on cytokine expression in monocytic cells. The step of PC was performed to amplify the cDNA copied from the mRNA of both the genes used, the gene of interest ((BCR/ABL) and the reference gene (GAPDH).

The PCR for GAPDH (housekeeping gene) was run for 24 hours and it was set up by mixing the 120 µL TaqMan® fast universal master mix (2X), 12 µL TaqMan® gene expression assay GAPDH (20x) and 60 µL Nuclease free water in a tube. Then the PCR for BCR/ABL PCR (gene of interest) was also kept running for 24h reactions it was prepared by mixing same reacgents except 12 µL TaqMan® gene assay BCR/ABL(20X) instead of TaqMan® gene expression assay GAPDH. Now the , 2 µL cDNA from each sample and the control were added three times to 48 well of 96 well plate fast RT-PCR, then 8 µL GAPDH PCR cocktail was put to wells A1-B9 and 8 µL BCR/ABL PCR cocktail was also added to wells C1-D9 (as shown below). Finally, a brief centrifuge was done to the plate and then the samples were run on the RT-machine and the results observed are as follows:

Result and Discussion:

The result for the experiment was unsuccessful when performed in the laboratory. The CT value for every cycle was calculated for each sample by the Real time software. The Table 2 represents the CT values for all the three samples listed in the experiment. Every sample was inserted three times for each housekeeping genes (GAPDH) and the gene of interest (BCR/ABL) from each of the RNA extraction method A and method B.

1

Table 2: The table represents the CT values for all three samples.

Table 3: Average and the standard error of the CT value of all three patients for both housekeeping gene (GAPDH) and GOI (BCR/ABL), also shows the ΔCt value and 2-ΔCt value.

Figure 1 The ΔCt values are displayed in the figure for all the patient samples to draw a comparison between the two methods used for RNA extraction and to determine the efficient method.

This graph represents the ∆CT value for patient samples A, B, C as well as the negative RT sample. The column blue signifies the ∆CT value obtained from extraction method A (crude analysis) for patient samples A, B and C, while the red column shows the ∆CT from extraction method B (trizol reagent) for patient samples A, B and C.

The extra column represents the RT-ve sample. The findings in this figure explain that the ∆CT form RNA extraction method A for both patients A, which is 9.35 and patient C, 7.4 is higher than RNA extraction method B which recorded 7.87 and 6.64 respectively for the same patients. In contrast, the ∆CT from RNA extraction method A for patient B which is 6.0 is much lower than method B which is 11.34.

The research was perfomed to compare the lysis of cells when they are treated with the DNAse method, Trizol reagent method for the extraction of RNA using the RT-PCR. It was also conducted to perform which method is more efficient and will generate high quality of RNA. The investigation helped to study the patient samples using BCR/ABL gene expressions.

The Real time is considered as a good tool in quantifying the data analysis for mRNA quantification.(Nolan, Hands and Bustin 2006).

On the basis of the results observed during the experiment we have concluded that the Trizol reagent methods have given more efficient results than the crude lysis method. Our results were also supported by the lower Ct values which indicates the high amount of RNA present in the samples (Dorak 2006)

As a result, the ∆Ct value of RNA extraction method B for patient A is 7.878 and patient C is 6.642, which is lower than the ∆CT value of method A for the same patients, which are 9.358 and 7.409 respectively (see figure1). However, the ∆CT value for RNA extraction method B for patient B showed the opposite, and the ∆Ct level of method B is higher than ∆Ct value of method A, which means low RNA is found in the sample. This inconsistency result in patient sample B is likely because the trizol reagent is sensitive method and needs high accuracy during its preparation steps. Little contamination was found which is indicated in the RT negative sample observed in Figure 1. This sample was used as the control and therefore no amplification can be expected in this sample but due to the contamination the amplification was seen. These results also prove that the RNA samples are very sensitive to any contamination therefore additional care must be provided during RNA isolation by trizol reagent to avoid contamination with RNases, which is more likely to occur during its preparation (Santella 2006).

Based on this, the result of this experiment is more likely to have been affected by the contamination either by genomic DNA or by RNase during the RNA extraction steps.

As a result of occurrence of the contamination, the ∆∆Ct in this experiment is affected; therefore, it is unable to calculate the value of ∆∆Ct and the fold change of BCR/ABL gene. Kurar et al. (2010) observed that, to measure the level of gene expression, it is compulsory to have RNA with good quality and quantity.

Moreover, the RNA must also be free of genomic DNA; otherwise, the end product of the RT-qPCR will be affected negatively. Also, using unclean or bad-quality RNA might inhibit the efficiency of RT-PCR, leading to negative results.

However, given the errors noted in this experiment, further corroborative investigations are needed before definitive conclusions can be reached about the expression level of BCR/ABL in patient who affected with chronic myeloid leukemia. It is highly recommended to repeat this experiment under controlled conditions to avoid the contamination and to achieve completely correct results.

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