316 report draft

The Hong Kong Polytechnic UniversityDepartment of Applied Biology and Chemical Technology

ABCT 316 Experimental Approach in Molecular Biology and Biochemistry

2011-2012 Semester 2

Project Report

GPR Activity Assay

Name: Tsoi Man Fung

Student ID: 10044509D

Group: 1

Group-mate: Tang Wai Man (10525360D)

Tsoi Man Fung 10044509D

Content

Content 2

Objective 3

Background Information 3

Materials and method 6

Detailed Procedures 11

Precautions 19

Results 20

Discussion 32

Further experiments 40

Reference 41

ABCT 316 Experimental Approach in Molecular Biology and Biochemistry Project Report


ObjectivesThe objective of this project is to test the promoter activity of Glucose-Regulated Protein 78kDa (GRP78) promoter in 293T/17 mammalian cells by using dual luciferase reporter system. This involves cutting of GRP promoter sequence in pGL3-GRP, inserting the GRP promoter sequence into pGL4 vectors, transfecting cells with recombinant plasmid vectors and test the promoter activity in the presence of drug treatment.

Background Information

(1) GRP78

GRP refers to glucose-regulated protein in endoplasmic reticulum. It is regarded as a chaperon that is used to correct protein misfolding. It is aimed at maintaining cellular homeostasis, preventing from entering cell apotosis. GRP78 can be activated by glucose starvation, unfold protein response (UPR) or endoplasmic reticulum stress. GRP protects more against stresscaused by depletion of ER Ca2+ than by a blockage in protein glycosylation. [3]

Activation of transcription of GRP genes is caused by endoplasmic reticulum stress which is medicated by the ERSE of GRP gene promoters and increase in level of GRP protein subsequently. Based on the mechanism, suppression of GRP genes in cells with ER stress increases cytotoxicity and results in cell apotosis. [26]

GRP78 promotes tumor proliferation, survival, metastasis and immunotherapy and it is an important biomarker for ER stress. In contrast, activation of GRP78 promoter could be used to drive therapeutic gene expression for gene therapy of some tumors. Besides, the combination of use of GRP78 promoter, photodynamic therapy could control expression of therapeutic gene temporally and spatially. [26]

The transcription efficiency of GRP78 is important for GRP78 gene expression and promoter strength are affected by GRP78 promoter activity. The promoter activity of GRP78 is investigated by using pGL4 vectors in this project.

Figure 1 shows the DNA sequence of GRP78 promoter. The underlined (-19 to -25 basepair upstream) is the TATA box.



(2) Luciferase Reporter Vectors

Luciferase is a reporter gene for transfection. This can link the cis-acting elements of a gene from gene interested to an unrelated gene to estimate induction results in change in gene expression due to gene regulatory sequence. This can be achieved by measuring the amount of product of reporter gene. It measures the RNA level indirectly by measuring level of protein.

A good reporter gene should contain gene which encodes for a protein originally absent from the host. The assay should be fast and sensitive. The assay should have broad linear range for analysis large and small change in promoter activity. Also, it should have no endogenous activity inside the cells. Firefly luciferase system was chosen due to its high specific activity, absence of endogenous activity and broad dynamic range, as well as convenient assay.

(3) E. Coli as component cellsE. Coli has a high growth rate. It can be used to amplify the amount of pGL4-GRP vectors. The OD600 should be between 0.4 and 0.45 so that the cells are grown at log phase or mid-log phase in order to provide high transformation efficiency. Moreover, value of OD600 about 0.4 indicates that the number of viable cells is not over 108 cells / ml culture. This could ensure that the culture does not grow to a higher density and minimize the appearance of ampicillin-sensitive satellite colonies. OD600 of 0.9 indicates the E.Coli strand has grown in late-log phase, which is another desirable condition for DNA transformation. It is however, provides a short time for collection cells. Due to low nutrient content in medium which could result in decrease in cell death, the cells should be stored at -80oC and incubated in medium with 15% volume per volume glycerol-containing medium to achieve maintenance of viability of cells.

(4) 293T cells [23]293T cells are regarded as human embryonic kidney stem cells. This type of cells allows the post-translational modifications for studying gene expressions in human cells. The “T” in 293T cells is SV40 large T antigen which is the major product of SV40 early viral region that causes the cells becomes malignant. It does not affected by plasmids and relative insensitive to toxicity by plasmids. This provides the condition of studying the promoter strength of GRP78 promoter in mammalian cells. Also, it adheres plastic surface to grow. This allows conducting cell lysis and washing in an easier way.

(5) Transient transfectionTransient transfection is a transfection that generates the unstable transfection. Transfected cells cannot have plasmids replicated to the next generation during mitosis. The expression of



gene is conducted within 48-72 hours for mammalian cells.

(6) Dual Luciferase Report assay

Dual luciferase system is used to eliminate the effect of different transfection efficiencies on luciferase activity. By performing co-transfection in transient transfection of two reporter genes, the internal control is used for normalization and minimize the inherent variability and transfection efficiency. [7] The internal control should have constant rate of gene expression for having reference. This reporter gene expression does not change under disturbance. The experimental reporter can be used to examine the promoter strength. This type of experimental reporter should contain multiple cloning sites. This allows the insertion of promoter of GRP.

Dual Luciferase Assay System integrates Firefly and Renilla Luciferase Assays within 1 test. It is used to examine the activity of GRP promoter.

Firefly LuciferaseFirefly Luciferase is a protein targeted to peroxisomes in all organisms. It does not required post-translational modification, thus it can be used as a reporter immediately. The reaction of Firefly luciferase is described in figure 2(A). It catalyze the reaction between beetle luciferin, oxygen and ATP to form oxyluciferin, AMP, pyrophosphate, carbon dioxide and light at 562nm. [16]

Promega’s system has included coenzyme A to enhance the kinetics of the reaction, thus sensitivity of the system improved and sustained light can be emitted which is long wnough for accurate detection [19]

Renilla luciferaseRenilla luciferase is protein found in Renillareniformis. No post-translational modification is needed thus it can be used as reporter gene after translation. It catalyzes reaction between coelnterazine and oxygen to form coelenteramide. Luminescence decay slowly that can be measured by luminometer in Promega’s system. [19]



(A)

(B)

Figure 2 shows the reactions of different luciferases. (A) shows reaction catalyzed by Firefly luciferase while (B) shows shows reaction catalyzed by Renilla luciferase

Materials and MethodsA. Method

The experiment started by performing double restriction digestion of both pGL4-10 and pGL3-GRP plasmid. The success of restriction digestion was confirmed by gel electrophoresis. The target DNA was then be purified from the gel. It is followed by ligation reaction to incorporate GRP promoter into pGL4-10 plasmid. Competent cells would be prepared at the same time. The competent cells prepared would be used to perform transformation. Clone screening would be done to pick and culture colonies.

The presence of GRP promoter sequence in transformed competent cells was confirmed by DNA extraction, double restriction digestion and gel electrophoresis. The plasmids extracted would be transfected into mammalian cells for studying promoter strength using in the presence of drug treatment. The promoter strength would be studied by using dual luciferase assay.

In order to reduce variability in transfection efficiency, pGL4-74 plasmid with specific plasmid are co-transfected into mammalian cells. pGL4-74 plasmid acts as an internal control that contains constitutive promoter for having comparison for having normalization to increase statistical significance and data consistency.



B. Materials(1) pGL4 plasmids from promega

pGL4-10 is used as vector for GRP and negative control for transfection to check if there is any leaky expression of genes. pGL4-13 and pGL4-23 are also used as positive control. pGL4-74 is the vector for normalization to minimize inherent variability. pGL4-10, pGL4-23 and pGL4-23 contain luc2 gene, which encodes for Firefly luciferase while pGL4-74 contains hRluc gene that encodes for Renilla luciferase.

pGL4-10 does not contain promoter sequence but it contains a multiple-cloning sites for insertion of promoter sequence for studying promoter strength of GRP in this project. pGL4-13 contains SV40 strong promoter that the expected result of this gene expression is high, resulting in high luciferase activity while pGL4-23 is to provide minimal expression as a reference. [5, 19]

(A)

(B)



(C)

(D)Figure 3 shows the vector map of (A) pGL4-10 (B) pGL4-13 (C) pGL4-23 and (D) pGL4-74 vector. The underlined words are promters present in vectors.

(2) Restriction EnzymesXhoI and BglII are used in experiment since recognition sequences of these two enzymes are found in the multiple cloning site of pGL4-10 and pGL3-GRP plasmids. These two enzymes have the same optium temperature of 37oC for performing double restriction digestion in optimal condition. Also, both enzymes generate sticky ends so that ligation reaction can be performed at a faster way and without additional steps, for example, addition of adapter. [9, 10]

(A) (B)Figure 3 shows the recognition sequence and site of cleavage of (A) XhoI and (B) BglII respectively.

(3) Gel Red Gel Red is an fluorescent molecule. It is non-carcinogenic compared to traditional fluorescent molecule ethidium bromide. It interchelates between DNA molecules to give fluorescent under presence of UV exposure. [11]



(4) DNA markersDNA markers are used for calibration to calculate the size of DNA fragments in the gel. Since the size of DNA fragments are between 200bp and 5000bp, 2 DNA markers are needed. Takara Biotechnology 100 bp DNA Ladder (Dye Plus), Bio-rad 100bp marker and Promega 1kb ladder

were used in this series of experiments.

(5) Wizard SC Gel and PCR clean-up systemTarget DNA fragments separated from gel electrophoresis are purified using this system. The working principle of this system is under presence of chaotrophic salts, DNA molecules binds to silica membrane. The DNA fragments are separated by dissolving target DNA from the gel into membrane binding solution which has guanidine isothiocyanate. Also, relative high percentage of recovery of DNA fragment at 200bp and 4000bp is more suitable in this experiment. [12]

(6) T4 DNA ligaseT4 DNA ligase is encoded by gene 30 of bacteriophage T4. It can be used to perform ligation under presence of ATP. It can perform ligation with sticky ends, which is suitable for this experiment. Cofactor required is ATP which is better than use of DPN (NAD) for the experiment. [16]

Figure 4 shows the example of catalytic reaction of T4 DNA ligase in sticky end.

(7) QIAprep Spin Miniprep KitDNA from E.coli is extracted and purified by this kit. It can reduce steps concerned about loose resin or slurries by silica membrane technology. No further process are needed for DNA purified. The products purified are in high quality. [17]



(8) Dulbecco’s Modifies Eagle Medium (DMEM)DMEM improves the formula of Basal Medium Eagle by increasing concentration of amino acids and vitamins 4 times. It can support 293T cells in this experiment.

(9) Fetal Bovine SerumThis medium contains essential growth factor for cell proliferation. It can be used for protection against chemical change such as change in pH, endotoxin. Fetal Bovine serum is used to provide complete medium for mammalian cell growth.

(10) Phosphate Buffer SalineThis buffer helps to maintain constant pH. This buffer is isotonic and non-toxic to cells that can maintain integrity of physiological status. It can be used to remove DMEM after culturing as the passive lysis buffer used are diluted by this buffer.

(11) AmpicillinAmpicillin with 100μg/mL can be used for clone screening. Transformed E.Coli contain ampicillin resistant gene, non-transformed cells would not grow in this medium. This can be used to select transformed cells for further experimental procedures in effective and easy way.

(12) LB mediumLB medium is used for growing of bacteria at liquid medium. It contains essential nutrients for bacteria to grow. This media can be used to incubate and select transformed E.coli under the presence of ampicillin in the medium.

(12) Genejuice Transfection Reagent for mammalian cellsThis reagent is used for transfection. The formula is optimized to achieve maximum transfection efficiency and minimize cytotoxicity. This can provide a better performance in transient transfection of 293T mammalian cells in the experiment. [18]

(13) Dithiothreitol (DTT)This is an reducing agent which can induce the reductive stress and also unfold protein response which triggers the endoplasmic reticulum stress.


Diagram showing the molecular structure of DTT


Detailed Procedures(1) Double restriction Digestion

Double restriction digestion of pGL4-10 and pGL3-GRP plasmids were performed according to the table shown in table 1 and table 2 respectively. The reaction mixtures were incubated at 37oC for 2 hours followed by storage at -20oC for storage.

Table 1. Volume required for DNA samples to be added to restriction digestion setup

Sample pGL3-GRP pGL4-10

Concentration 624.5 ng/μL 198.5 ng/μL

Amount of DNA required (μg) 4 1

Volume of sample required 6.4 5

Table 2. Experimental Setup for double restriction digestion for pGL3-GRP and pGL4-10 plasmid

SubstancesTube Adding

sequence1 (pGL3-GRP) 2 (pGL4-10)

Volume of

substances added (μL)

pGL3-GRP 6.4 03

pGL4-10 0 5

XhoI 1 0.5 4

BglII 1 0.5 5

10x Buffer 2 2 2

ddH2O 9.6 12 1

Total volume 20 20

(2) Preparation of Luria-Bertani (LB) brothLB broth was prepared by weighting 10g LB powder and dissolving the powder in 500mL ddH2O. The powder then was mixed well and autoclaved to kill microorganisms in the medium.

(3) Preparation of agar plateThe agar plates were prepared by cooling the molten agar to about 50 to 60oC. 500μL of ampicillin stock with concentration of 100μg/μL were added to the molten agar to adjust the concentration of ampicillin in molten agar of 100μg/mL. The molten agar was then mixed by swirling and poured to 5 petri dishes with 30-35 mL of molten agar in each plate. The petri dishes were cooled at room temperature and allowed them to set. All the plates were inverted after they had hardened completely and stored at 4°C until needed.



(4) DNA electrophoresis from product of double restriction digestionThe gel was prepared by weighting 0.509g agrose and put into 50mL 1X TAE buffer. The mixture was put into microwave oven to melt agrose in the oven. 5μL was then added to the molten agrose. The gel was casted and comb was removed when the gel was set. The gel was placed inside the gel tank and the gel tank was filled with 1X TAE buffer.Each reaction product was spinned down and added 4μL loading dye. Both positive controls were prepared by mixing 1μL DNA, 1μL 6X buffer and 4μL ddH2O. Positive Controls, samples and DNA markers were loaded according to the table 3.

Table 3. Experimental setup for gel electrophoresis in week 2

Lane 1 2 3 4 5 6

Substance 1kb ladder*1 100 bp ladder*2

pGL4-10(restriction digestion product)

pGL3-GRP(restriction digestion product)

pGL4-10(positive control)

pGL3-GRP(positive Control)

Volume of substance loaded (μL)

5 5 24 24 6 6

Note: *1 The 1 kb ladder is manufactured by Bio-Rad®.*2 The 100bp ladder is manufactured by Promega®

After all samples were loaded, gel electrophoresis was performed at 100V at constant voltage for 40 minutes. 4 Eppendorf tubes were preweighted and marked with respective weight. The gel electrophoresis was stopped when the gel front was about 1/3 from bottom of the gel. Gel image was captured and bands were identified. The gel segments were sliced by sterile razor under UV illumination. Weight of each slice was determined by using balance and pre-weighted Eppendorf tubes as reference.



(5) DNA purification from gel electrophoresisEach tube was added with 10μL of membrane binding solution per 10mg of slice of gel. Detailed volume of membrane binding solution had been shown in the table 4. Each tube was vortexed and incubated at 65oC until gel slice was dissolved completely.

Table 4. Experimental setup for addition of membrane binding solution to each Eppendorf tube

Tube 1 2

Sample pGL4-10 GRP

Mass of gel (g) 0.1539 0.1649

Volume of membrane binding solution added (μL) 154 165

SV Minicolumn was inserted into collection tube. Each gel mixture was transferred to Minicolumn and incubated at room temperature for 1 minute. Each tube was centrifuged at 13000rpm for 1 minute and flow-through was discarded. Each Minicolumn was inserted back to their collection tubes respectively. 700μL of membrane wash solution was added to the column and followed by performing centrifugation at 13000rpm for 1 minute. Flow through was discarded and columns were reinserted to their respective column. The step was repeated by applying 500μL of membrane wash solution and centrifuged at same speed for 5 minutes. The collecting tubes were re-emptied and recentrifuged for 1 minute.

Each minicolumn was transferred to 1.5mL Eppendorf tube and 50μL of ddH2O was applied to each tube. The tubes were incubated at room temperature for 1 minute. All tube were followed by applying centrifugation at 13000rpm for 1 minute. Minicolumns were discarded and stored at -20oC.

(6) Purified DNA concentration determinationDNA concentrations were determined by applying 2μL of sample into specific well of machine using ddH2O as a reference.



(7) LigationLigation reactions were performed according to the table 5. Tube 2 and 3 are used as negative control for ligation reaction.

Table 5. Experimental setup for Ligation reaction

TubeAdding

sequence1 2 3

Tube content pGL4-10 + GRP No insert (no GRP) No ligase

Volume of

substance to be added (μL)

pGL4-10 2 2 2 3

GRP 2 0 2 4

ddH2O 3.5 5.5 5 1

T4 ligase 1.5 1.5 0 5

Buffer 1 1 1 2

Each tube was incubated for 2 hour at room temperature and stored at -20oC afterwards.

(8) Preparation of Competent cells5 mL E. coli culture was grownb in LB medium. 1mL of culture was added in 100mL LB medium and grown overnight. The culture was grown to OD600 equals 0.4-0.5 by start collecting at 0.35. Cultures were transferred to 50mL centrifuge tube and chilled on ice for 10 minutes.

Each tube was centrifuged at 4100rpm at 4oC for 10 minutes. It is followed by discarding medium and re-suspending cells in 30mL ice-cooled 0.1M calcium chloride solution. The re-suspended culture was centrifuged at 4100rpm for 10 minutes. The medium was discarded and the pellet formed was re-suspended with 2mL ice cooled 0.1M calcium chloride with 15% glycerol. Each 200μL aliquot was transferred to new Eppendorf tubes and stored at -80oC.



(9) Transformation5 tubes of competent cells were used for transformation. Each tube is prepared according to the table below.

Table 6. Experimental setup for transformation

Tube Sample to be added Volume of sample added (μL)

1 Intact pGL4-10 5

2 pGL4-10 without insert in ligation reaction tube 2 5

3 pGL4-10 without ligase in ligation reaction tube 3 5

4 pGL4-GRP (ligation reaction tube 1) 5

5 ddH2O 5

Each reaction tube was chilled on ice for 10 minutes. It is then transferred to 42oC for 45 seconds and returned to 4oC immediately after transferring to 42oC. Each tube was added with 800μL medium (without ampicllin). All tubes are incubate at 37oC, 220rpm for 45minutes. For each tube, 50μL of tem are extracted and spread on each agar plate prepared in week 1 respectively. All agar plates were left to dry and inverted afterwards. All plates are incubated at 37oC.

(10) Clone Screening4 colonies were picked up in plate of pGL4-GRP. Each colony was transferred to centrifuge tube with 5mL LB medium with ampicillin concentration of 100μg / mL. Each tube was incubated overnight with shaking. After overnight shaking, all tubes were performed centrifuge to form pellet and medium were discarded.

(11) DNA extraction, purification and concentration determinationPellets from clone screening were re-suspended in 250μL buffer P1 for each tube. 250μL of buffer P2 was added to each tube and mixed thoroughly by inverting each tube 4-6 times. 350μL of buffer N3 was added to each tube and mixed thoroughly by inverting each tube 8-10 times. Each tube was centrifuged for 10 minutes at 13,000 rpm. Supernatant of each tube was applied to QIAprep column and centrifuged for 1 minute at same speed. Flow through was discarded and washed with 0.75mL buffer PE. Each tube was centrifuged at same speed for 1 minute. Flow through was discarded and the tube was centrifuged for 1 minute. Each column was transferred to a new Eppendorf tube and 500μL of ddH2O was added to the column and standed for 1 minute. Each tube was centrifuged for 1 minute. The DNA concentration was determined.



(12) Double Restriction Digestion for purified DNA samples4 Eppendorf tubes were prepared for double restriction digestion. The restriction digestion reaction for each tube was setup according to the table shown below.

Table 7 Experimental setup for second double restriction digestion

Substance Volume of substance added (μL) Adding sequence

Purified DNA 4 3

XhoI 0.5 4

BglII 0.5 5

10x buffer 2 2

ddH2O 13 1

Each tube was incubated at 37oC for 2 hours. The tubes were stored at -20oC for storage afterwards.

(13) Cell passageThe confluent cells were washed with phosphate buffer saline. The cells were then treated with trypsin to detach cells by breaking down the extracellular matrix. The cells were incubated with DMEM and Foetal Bovine Serum for inactivating trypsin.

(14) Setup gel for gel electrophoresis.The gel was prepared by mixing 0.527g apgrose with 50mL 1x TAE buffer in a conical flask. The flask was put to microwave oven to melt agrose. 5μL of Gel Red was added to the molten agrose. The gel was casted and the comb was removed.



(15) Gel electrophoresis for product of double restriction digestion For each double restriction digestion setup in session (12), 4μL of DNA loading dye was added to each tube. Samples were loaded according to table shown below.

Table 8 Experimental setup for second gel electrophoresis

Lane number Substance Volume of substance loaded (μL)

1 DNA ladder (1kb) 5

2 Restriction digestion setup tube 1 24




6 DNA ladder (100bp) 5

Gel electrophoresis was performed at 100V in constant voltage for 40 minutes.

(16) TransfectionTransfection is performed by preparing 6 new Eppendorf tubes. For each tube, 3μL Genejuice and 80μL was added. DNA samples and ddH2O were added to each tube according to the following table below.

Table 8 Experimental setup for second gel electrophoresis

Tube Substance added Volume of substance added (μL)

1ddH2O 4

pGL4-74 1.6

2pGL4-GRP 4

pGL4-74 1.6

3pGL4-10 4

pGL4-74 1.6

4pGL4-13 5.2

pGL4-74 1.6

5pGL4-23 4

pGL4-74 1.6

6 ddH2O 5.6

All tubes are incubated at room temperature for 15 minutes. Mixtures of each tube were added into each well according the diagram below. Each well was added with 22.1μL of Genejuice mixture according the diagram . The 24-well plate was put into 37oC incubator for



24 hours.

Figure 5 shows the detailed experimental setup of transfection, drug treatment in 24-well plate. The number in the circles is the number to be indicated tube number in cell lysis and dual luciferase assay.

(17) Drug treatment1μL of DTT or ddH2O was added to each well according to diagram .

(18) Cell lysisGrowth medium of each well was removed. Each well was rinsed with 200μL PBS buffer twice. 1x PLB was prepared by mixing 500μL of 5x PLB with 2000μL of PBS. Each well was dispense with 100μL passive lysis buffer. The plate was shaked on orbital shaker for 15 minutes. Lysate from each tube was transferred into a new Eppendorf tube respectively and labeled according to numbers in diagram .

(19) Determination of promoter strength by using dual luciferase reporter assay24 new Eppendorf tubes are prepared by adding 10μL cell lysate in last session. 35μL of luciferase assay reagent II was dispensed into each microcentrifuge tube and mixed but pipetting. The first reading was taken after mixing. 35μL of Stop and GLO reagent was added and mixed by pipetting. The second reading was taken.



Precautions

There are several precautions in the experimental procedures.For collection of competent cells, it should be collected at OD600 of 0.35 so that the comptent cells are within early or mid-log phase.

For DNA purification after clone screening, the RNAase A should be put in ice to prevent degradation of enzyme. Buffer P2 should be closed immediately after extracting known volume of P2 to avoid acidification of buffer. ddH2O for elution is dispensed on the centre of QI prep membrane to optimize elution of DNA and ddH2O for elution is within pH 7.0-8.5 to achieve maximal elution efficiency.

For transfection, the Genejuice transfection reagent should be mixed with DNA in the absence of serum since addition of serum would hinder the formation of complex and resulting in decrease in transfection efficiency.

For dual luciferase reporter assay, the luciferase assay reagent II and Stop and GLO reagent are light sensitive that should be covered with aluminium foil.



Results(1) DNA electrophoresis from product of double restriction digestion

Figure 6 shows the gel photo of first gel electrophoresis. The samples to be loaded in lanes are (1) 100bp marker from Bio-Rad, (2) 1kb marker from Promega, (3) double restriction digestion products in pGL4-10, (4) double restriction digestion products in pGL3-GRP, (5) intact pGL4-10 plasmids, (6) intact pGL3-GRP respectively

2 2.2 2.4 2.6 2.8 3 3.20.5

0.550.6

0.650.7

0.750.8

0.850.9

0.95

f(x) = − 0.427937796648728 x + 1.9005293928338R² = 0.986949390622113

Standard curve of 100bp DNA marker

log(Molecular weight)

Rf

(A)



2 2.5 3 3.5 4 4.50

0.10.20.30.40.50.60.70.80.9

1

f(x) = − 0.414305473669903 x + 1.85683526076843R² = 0.991383636767025

Standard curve of 1kb DNA marker

log(Molecular weight)

Rf v

alue

(B)

Figure 7 shows the standard curve of logarithm of DNA molecular weight against Rf value using (A) 100bp marker from Bio-Rad, (B) 1kb marker from Promega.

Table 9. Calculated size DNA fragments in different lanes by standard curve

Lane BandDistance from

origin (cm)Rf value

Log (MW) calculated

Calculated MW (bp)

Expected MW (bp)

Comment

3 a 2.26 0.3319 3.65 4466 4197 pGL4-10

4b 2.27 0.3333 3.68 4775 4804 pGL3

c 6.42 0.9427 2.24 173 146 GRP

5d 1.94 0.2849 3.79 6225

Nicked / relaxed pGL4-10 plasmid

e 3.00 0.4405 3.42 2621Supercoiled

pGL4-10 plasmid

6

F 1.52 0.2232 3.94 8771Relaxed pGL3-

GRP

G 2.14 0.3142 3.72 5288 Nicked pGL3-GRP

H 2.95 0.4332 3.44 2370Supercoiled pGL3-GRP



(2) DNA concentration determination for purification of DNA

Table 10. DNA concentrations and parameters of DNA purified for pGL4-10 and GRP

Sample pGL4 - 10 GRP

A260 0.4 0.252

A280 0.291 0.262

A320 1.118 0.165

A260 / A280 1.607 0.897

A260 / A230 0.818 0.212

DNA Concentration (ng / μL) 13.9 4.4

Volume obtained from purification (μL) 50 50

DNA obtained from purification (ng) 695 220

(3) Transformation

Figure 8 shows the results of transformation of E.coli for ligation reaction products. The number of colonies in each plate are shown in the right hand corner.



From the above diagram, transformation efficiency can be calculated by using transformation efficiency.

Table 11 Transformation efficiency of different plates in transformation reation

Transformation reaction No. of colonies DNA spread (ng)Transformation efficiency(Transformants / ng DNA)

pGL4-10-GRP 944 0.91 1037

Intact pGL4-10 (+ve control) 2720 62.31 44

No insert (-ve control) 4 0.28 14

No ligase (-ve control) 0 0.22 0

(4) DNA cloning and screening from transformed E. coli cells

Table 12. DNA concentrations and parameters of DNA purified from DNA cloning and screening of transformed E.coli cells

Tube 1 2 3 4

Presence of pellet Yes Yes Yes YesOD230 2.34 2.32 2.31 2.05OD260 5.03 5.05 5.02 4.35OD280 2.81 2.83 2.65 2.16OD320 0.195 0.248 -0.043 -0.57OD260

OD2801.847 1.86 1.881 1.802

OD260

OD2302.251 2.319 2.153 1.878

Concentration (ng / μL) 242 240 253 246



(5) Gel electrophoresis for product of double restriction digestion

Figure 9(a) showing the image of gel electrophoresis. Lanes arrangement are (1) 1kb DNA ladder, (2) Double restriction digestion product from tube 1, (3) Double restriction digestion product from tube 2, (4) Double restriction product from tube 3, (5) Double restriction digestion product from tube 4, (6) 100bp DNA ladder respectively.

2 2.2 2.4 2.6 2.8 3 3.2 3.40.5

0.550.6

0.650.7

0.750.8

0.850.9

0.95

f(x) = − 0.266362476695642 x + 1.45282925967872R² = 0.99456472886668

Standard curve of 100 bp ladder

log(MW)

Rf v

alue

(A)



2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.20.20.30.40.50.60.70.80.9

f(x) = − 0.348259725482544 x + 1.65465039386897R² = 0.998315178115158

Standard curve of 1kb ladder

log(MW)

Rf v

alue

(B)

Figure 10 shows the standard curve of logarithm of DNA molecular weight against Rf value using (A) Takara Biotechnology 100 bp DNA Ladder (Dye Plus), (B) 1kb marker from Promega.

Table 13. Calculated size of DNA fragments in gel electrophoresis using standard curve

Lane Band MW (bp) log (MW) Migration distance (cm) Rf

2A 4107.147973 3.61354 1.83 0.396104

B 151.0341581 2.179075 4.03 0.872294

3C 4048.795797 3.607326 1.84 0.398268

D 145.4873263 2.162825 4.05 0.876623

4E 4107.147973 3.61354 1.83 0.396104

F 148.2347997 2.17095 4.04 0.874459

5G 3934.566777 3.594897 1.86 0.402597

H 140.1442057 2.146575 4.07 0.880952

(6) Cell passage

(A) (B)Figure 11 shows the 293T mammalian cells (A) before (100x) and (B) after treatment of



trypsin (40x)(7) Determination of promoter strength by using dual luciferase reporter assay

Table 14.Raw data of results on dual luciferase reporter assay

tube Sample Treatment 1st reading 2nd reading ratio

1ddH2O

+pGL4-74

ddH2O90 27404

N/A2 94 2928

3DTT

87 5573

4 74 4624

5pGL4-GRP

+pGL4-74

ddH2O1821 425 4.2847

6 3056 147 20.7891

7DTT

412 98 4.2041

8 307 115 2.6696

9pGL4-10

+pGL4-74

ddH2O91 143 0.6364

10 63 109 0.5780

11DTT

77 119 0.6471

12 87 121 0.7190

13pGL4-13

+pGL4-74

ddH2O937 123 7.6179

14 22055 181 121.8508

15DTT

13378 171 78.2339

16 1459 103 14.1651

17pGL4-23

+pGL4-74

ddH2O90 135 0.6667

18 87 122 0.7131

19DTT

107 132 0.8106

20 91 124 0.7339

21 ddH2O ddH2O 87 129 N/A

22 91 108

23DTT

72 113

24 97 92

Data treatment:The corrected reading should be calculated according to the formula below.



The corrected ratio is calculated by using formula below.

The ratio is normalized according to the equation below

Table 15.Raw dataCorrected data of results on dual luciferase reporter assay

tube Sample Treatment 1st reading 2nd reading

1

ddH2O + pGL4-74

ddH2O3.25 27293.5

2 7.25 2817.5

3DTT

0.25 5462.5

4 -12.75 4513.5

5

pGL4-GRP + pGL4-74

ddH2O1734.25 314.5

6 2969.25 36.5

7DTT

325.25 -12.5

8 220.25 4.5

9

pGL4-10 + pGL4-74

ddH2O4.25 32.5

10 -23.75 -1.5

11DTT

-9.75 8.5

12 0.25 10.5

13

pGL4-13 + pGL4-74

ddH2O850.25 12.5

14 21968.25 70.5

15DTT

13291.25 60.5

16 1372.25 -7.5

17

pGL4-23 + pGL4-74

ddH2O3.25 24.5

18 0.25 11.5

19DTT

20.25 21.5

20 4.25 13.5

Note: The highlighted data are considered to be eliminated as the corrected reading should not be negative



Table 16.Corrected and normalized data for dual luciferase reporter assay

Sample treatmentTube

Mean SEM1 2

pGL4-GRP + pGL4-74ddH2O 0.1270 1.8730 1 0.8730

DTT 1.1270 　 1.1270 0

pGL4-10 + pGL4-74ddH2O 0.0030 　 0.0030 0

DTT 0.0005 　 0.0005 0

pGL4-13 + pGL4-74ddH2O 1.5661 7.1746 4.3703 2.8042

DTT 5.0583 　 5.0583 0

pGL4-23 + pGL4-74ddH2O 0.0031 0.0005 0.0018 0.0013

DTT 0.0217 0.0072 0.0145 0.0072

Note: As some data is eliminated due to invalid range after data correction, SEM cannot be calculated.

Figure 12 shows the Corrected and Normalized ratio of different reporter vectors in mammalian cells under drug treatment



Since there some data eliminations, the data reliability cannot be treated using statistical calculation. The statistical treatment is done by using normalized ratio without data correction.

The normalized ratio is calculated by calculating the ratio by using the equation below.

The ratio is normalized according to the equation below

Table 17. Normalized data for dual luciferase reporter assay without data correction

Sample TreatmentExperimental setup

Mean SEM1 2

pGL4-GRP and pGL4-74DTT 0.3353 0.2129 0.2741 0.0612ddH2O 0.3418 1.6582 1 0.6582

pGL4-10 and pGL4-74DTT 0.0516 0.0574 0.0545 0.0029ddH2O 0.0508 0.0461 0.0484 0.0023



Based on the above results, the ANOVA test, Tukey’s test and two-tailed t test are carried out.

Table 18. Results ANOVA test calculationCorrection factor 21.3924Sum of square, total 116.7092Sum of square, judge 1.3489Sum of square, sample 61.2652Sum of square, error 54.0951Degree of freedom, total 15Degree of freedom, sample 7Degree of freedom, judgement 1Degree of freedom, error 7Mean square, sample 1.3489



Mean square, judgement 8.7522Mean square, error 7.7279F ratio, sample 0.1746F ratio, judge 1.1325

Table 19. Summary of ANOVA test

Source of variationDegree of freedom

Sum of square

Mean square

Variance ratioCritical Variance ratio (0.05)

Column (Samples) 7 61.2652 1.3489 0.174550207 3.78704354

Row (Judges) 1 1.3489 8.7522 1.132544862 5.59144778

Error 7 54.0951 7.7279Total 15 215.2590

p-value for samples: 0.983 > 0.05p-value for judges: 0.323 > 0.05

Conclusion for ANOVA test: There is no significant difference between samples as varience ratio is less than critical variance ratio

For Tukey’s test, the least significant difference is calculated by using 0.05 as level of significance.Standard Error (SE) 1.9657Least significant Difference (LSD) 11.0000

The Tukey’s test are calculated by using the equation belowValue in the box = Value of sample in the column – value of sample in row

Table 20. Results of Summary of Tukey’s test



From results of Tukey’s test, no samples have greater difference than the least significant difference. This shows that the results are insignificant.

For two tailed t-test, the null hypothesis of the test is that DTT treated pGL4-GRP gene expression has significant difference compared to ddH2O treated. The alternative hypothesis of the test is that DTT treated pGL4-GRP gene expression has no significant difference compared to ddH2O treated

Table 21 Test statistics of two-tailed t-test

Tube

Sample Treatment 1 2 Sum

pGL4-GRP and pGL4-74 DTT 0.3354 0.2129

pGL4-GRP and pGL4-74 ddH2O 0.3418 1.6582

Difference between 2 treatments -0.0064 -1.4453 -1.4517

Difference between 2 treatments2 4.1363E-05 2.0889 2.0889

Varience is calculated by using the formula below.

t-value is calculated using the formula below.



Table 22. Summary table for the values calculated in two-tailed t-test

s (varience) 1.017430736

t value -2.853712018

degree of freedom 1

p value 0.49717961

As p-value is less than 0.95, the null hypothesis is rejected that there is no significant difference between treatment of DTT and ddH2O.



Discussion(1) First gel electrophoresis, DNA purification from gel electrophoresis

In the first gel electrophoresis, lane

From the results of gel electrophoresis, there is one band (band a) in lane 3 which has molecular weight of 4197bp. It is similar to size pGL4-10. From the expected results, ideally, the gel photo should have 2 bands in lane 3. It is, however, due to very small size of one of the digested product (13bp), the digested fragments are ran out of the gel. The use of agrose gel in gel electrophoresis is not significant enough to check the size of the DNA fragments. Therefore, the success of double restriction digestion is not known in this stage. It can be known in DNA transformation.

In lane 4, there are two bands. Band b is a thick band with molecular weight of about 4800bp. There are several possible reasons for formation of this thick banding. The first one is the presence of undigested plasmids. The undigested plasmids have similar size compared to size of pGL3 vector. They may stay at much closed position and results in fusion of two bands. It is, however, to be noted that the amount of DNA samples applied is very large. The thick banding maybe due to high amount of DNA sample applied.

Band c in lane 4 appears to be much fainter. It shows that the purity of DNA is not enough or the double restriction digestion of plasmids does not have enough time. DNA concentration determination can further examine the purity of the GRP promoter.

In lane 5, band d is the nicked or relaxed plasmids while e is the supercoiled plasmids. When the plasmids are centrifuged for long time, the plasmid may form linear / relax plasmids which has a large surface area. It can pass through the gel matrix less efficiently under same electric field. The supercoiled plasmids has a more compact in size when has a lower surface area. The plasmids can pass through the gel matrix more easily under same electric field.

In lane 6, Band f is the nicked or relaxed circular plasmid. Band g is the linear plasmids Band H is the supercoiled plasmids. This is due to random coiling and the practices in DNA extraction and centrifugation mentioned above.

According to the gel image, there is a significant “smelling effect”. This is due to thickness of the agrose gel. When gel electrophoresis is performed. At the centre of gel, the heat dissipation is less significant than that in the edge. Molecules at the centre move faster compared to molecules near the edge of the gel due to higher kinetic energy.



Also, the gel was not set at proper position when the gel photo was taken. The

(2) DNA concentration determination from gel electrophoresisThe A260 is the relative absorbance for nucleic acid, this includes RNA and DNA. The A280 is the absorbance of protein due to presence of aromatic amino acids. A320 is the measurement of turbidity of solution which indicates the possible contaminations.

Ratio of A260 to A280 indicates the possibility of protein contamination. Based on the results, both GRP and pGL4-10 does not have value of close to 0.6, this indicates there is few protein contaminations. The high purity of DNA should have around 1.8-2.0. Both GRP and pGL4-10 has low purity. The value of GRP for A260 to A280 ratio is 0.82, this value shows the sample has very low purity.

A320 is the measurement of turbidity of the sample. The high turbidity in two samples maybe due to presence of DNA residue in the previous sample or organic compound in samples.

A260 to A230 is an indicator for contaminations by organic compounds. From the results, it shows that both GRP and pGL4-10 samples are contaminated by the organic compound. This is doe to presence of ethanol in samples during elution. This may also be due to machine contaminations or scratching of pipette tips on the machines.

There are some sources of errors. The measurement of DNA concentration was carried by using same pipette tip for measuring concentration of both pGL4-10 and GRP. Also, during calibration, the DNA concentration was 0.5ng /μL. No re-calibration was done to minimize the error.

(3) Ligation reactionFrom ligation reaction, there are two control setups. Two controls are the negative controls. Tube 2 (without GRP) is used to ensure that the success of double restriction digestion. The absence of GRP could not allow the self-ligation to occur as the two sticky ends are not complementary to each other. After transformation, if there are some colonies present, this indicates the problem(s) exist in double restriction digestion.

Tube 3 (without ligase) is used to check if the there are some uncut plasmids. In the absence of ligase, digested plasmids could not form circular plasmids. After transfeormation, if there are some re some colonies present, this indicates the problem(s) exist in double restriction digestion.



(4) Preparation of Competent cellsThe OD600 should be between 0.4 and 0.45 so that the cells are grown at log phase or mid-log phase in order to provide high transformation efficiency. Moreover, value of OD600 about 0.4 indicates that the number of viable cells is not over 108 cells / ml culture. This could ensure that the culture does not grow to a higher density and minimize the appearance of ampicillin-sensitive satellite colonies. OD600 of 0.9 indicates the E.Coli strand has grown in late-log phase, which is another desirable condition for DNA transformation. It is however, provides a short time for collection cells. Due to low nutrient content in medium which could result in decrease in cell death, the cells should be stored at -80oC and incubated in medium with 15% volume per volume glycerol-containing medium to achieve maintenance of viability of cells. [21, 22]

(5) Transformation

Plate 4 (intact pGL4-10) is used as a positive control for transformation. The formation of colonies on this plate indicates the success of transformation. E.coli cells contains the pGL4-10 plasmids. Therefore, they can transcribe and translate β-lactamase. If there is absence of colonies in plate 1, this indicates the problem existed in ligation reaction instead of transformation reaction.

Plate 1 (pGL4-10-GRP) contains the ligation reaction mixture. The plasmids are transformed into the E.Coli cells and gene encoding for β-lactamase is expressed and amplified to form colonies. Cells in each colony are genetically identically.

Plate 2 (no insert) is used to check for the success of double restriction digestion. The formation of colonies on this plate may due to several possible reasons. The first possible reason is the unspecific ligation reaction. Based on the recognition sequence in DNA, there are 2bp in recognition which is complementary to each other (figure ). It is however, the condition is not suitable for having this kind of ligation reaction. The second possible reason is the ligation between 2 plasmids. The complementary ends of plasmids can sticky ends and results in formation of plasmids. The formation of this type of plasmid is very low rate. The third possible reason is the problem in one of the double restriction digestion. During the double restriction digestion, there are some plasmids digested by one of two restriction enzymes. This forms a sticky end and T4 ligase can perform ligation reaction for forming circular plasmids.

5’ … C G A T C T … 3’3’ … T C T A G A … 5’

Plate 3 (no ligase) is used as a negative control to check for the success of restriction digestion



reaction. The absence of colonies indicates that there are no intact pGL4-10 plasmids in the mixture as pGL4-10 has ampicillin resistant gene.

Plate 5 (no vector) is used as a negative control to check the presence of antibiotic resistant E.coli or contaminations of medium by foreign strand of bacteria in the competent cells medium. The absence of colony indicates that the competent cells does not have gene encodes for β-lactamase.

The transformation efficiency of pGL4-10 is not as high as ligation reaction mixture which is deviated from the expected results. This may due to presence of growth-inhibiting substances secreted by E.coli killed by ampicillin in the agar plate which causes the increase in number of colonies is not in linear proportion with DNA amount.

Further experiments are needed for transformation in order to identify the cause of colony formation of setup without insert. This can be achieved by incubating pGL4-10 plasmids with XhoI and BglII respectively. Plasmids in different setup are purified by gel electrophoresis. The purified digested plasmids are transformed into E.coli respectively. If the plates contained colonies, this indicates that the problem of undigested plasmids in the sample.

(6) DNA cloning, screening and purificationFrom the DNA extraction, tube 1 to 4 have over 1.8 on ratio of A260/A280. This indicates that the DNA samples are pure enough. During purification, the concentration for each tube is around 240 to 250 ng /μL. The turbidity of the solution is insignificant for tube 1 and 2. The negative value of A320 in tube 3 sample can be neglected. It is however, the value of tube 4 is significantly negative. This might be due to machine error. The value of A260 / A230 indicates the possible contaminants of the sample. Value of all tubes is over 1.7, which is desirable for DNA sample.

(7) Second double restriction digestion and gel electrophoresisFrom the results of gel electrophoresis, there are two bands in all tubes. Band B, D, F and H have molecular weight of about 150bp, which is close to size of GRP promoter. This indicates the DNA extracted contains GRP promoter sequence. Band A, C, E and G have molecular weight of about 4000bp. This indicates the DNA extracted contains pGL4-10 vector. It has to be noted that band A, C, E and G has continuous band due to DNA degradation. The enzyme cuts at other regions, resulting non-specific digestion. Also, during storage with water under -20oC, the DNA molecules have DNA degradation.

(8) Transfection



Well 21 to 24 is a negative control. ddH2O and Genejuice added to the mixture is to ensure that the increase in luminometer reading is due to DNA transfection. Also, the values can be used as a reference to be substracted in reading of each well since the reading of luminometer is due to background noise.

Well 1 to 4 is a positive control of addition of ddH2O and pGL4-74 vector. This is used to show that the increase in second reading is due to transfection with pGL4-74 vector.

Well 9 to 12 is a positive control with addition of pGL4-74 and pGL4-10, these wells are used to check if there are any leaky expressions of pGL4-10 vector in the absence of promoter sequence. The increase in first reading is due to leaky expression of pGL4-10 vector in mammalian cells.

Well 13 to 16 is a positive control with addition of pGL4-13 and pGL4-10. pGL4-13 vector contains SV40 promoter which allows the maximal expression of Luc2 gene. This can be used as a reference of data validity of well 5 to 9.

Well 17 to 20 is a positive control with addition of pGL4-23 and pGL4-10. pGL4-23 vector contains minimal promoter which allows the minimall expression of Luc2 gene. This can be used as a reference for studying the expression level of Luc2 gene under the presence of GP promoter.

DTT is a reducing agent. It can disrupt the formation of disulphide bond[25]. This can induce reductive stress in endoplasmic reticulum of mammalian cells. It can be used to study the effect of ER stress on change of promoter strength.

For each group, 2 wells were treated with DTT while another 2 wells were treated with ddH2O. DTT is an reducing agent of containing thiol group. This reagent can induce reductive stress in endoplasmic reticulum. Treatment with DTT in two wells is used to study the effect of DTT on gene expressions of Luc2 gene in the absence of GRP promoter. If there is increase in first reading, it may be due to random error. The ddH2O treatment is used as a negative control.

Well 5 to 9 are the experimental setup. This can show that the promoter strength of GRP using pGL4-10 vector. The DTT treatment is used to induce ER stress. This can be used to study the effect of ER stress on gene expression of GRP78. The ddH2O treatment is used as a reference and a negative control to show that the change in first reading is due to presence of DTT in treatment.

(9) Dual Luciferase Assay



Well 21 to 24 are considered as negative controls, which have been applied ddH2O only in which these wells have no transfection reactions in these wells. The readings of these tubes are due to background signal, which is used as a set of reference value for comparison.

Well 1 to 4 have been added with ddH2O and pGL4-74 plasmids. The first reading of each well has no significant difference as no vectors containing Luc2 gene had been added into genejuice mixture during transfection. The second reading has increased significantly due to presence of pGL4-74 plasmids in mammalian cells, resulting gene expressions of Renilla luciferase.

Consider the overall experimental results in this session, the second readings of well 6 to 20 are close to that of negative control setup. This indicates that the transfection efficiencies of these wells are very low.

From the normalized ratio, results of well 5 to 20 have shown there are luciferase activity in well 5 to 20.

For well 13 to 16, pGL4-13 and pGL4-74 plasmids are added to the mammalian cells. This setup is used as a positive control. pGL4-13 plasmids contains SV40 promoter [5], providing maximal expression of Luc2 genes. With low Renilla Luciferase activity, the normalized ratio is high. This indicates the gene expression of Luc2 gene is high and the promoter strength of SV40 is the highest.

Well 17 to 20 are the positive control with minimal expression level. pGL4-23 plasmids has a minimal promoter for gene expression of Luc2 gene at minimal expression level. Therefore, the level of luciferase activity is minimal.

Well 9 to 12 are the positive control. Compare well 17 to 20, the normalized ratio of pGL4-10 transfected cells have higher Firefly luciferase activity than pGL4-23 transfected cells. This shows that gene expression of Luc2 gene pGL4-10 in mammalian cells. This is due to leaky expression of Luc2 gene pGL4-10 in mammalian cells.

Well 5 to 8 has been added pGL4-GRP and pGL4-74 plasmids. Compared to normalized ratio for well 9 to 12, the normalized ratio has increased in both DTT treated and ddH2O treated mammalian cells. This indicates the GRP promoter has increased the gene expression significantly as a gene regulatory sequence. The normalized ratio of both ddH2O treated is lower than that if DTT treated. The expected results are DTT treatment increases luciferase activity.



There is a possible reason. DTT treatment causes the unfold protein response. This increases the ER stress and activates transcription of GRP promoter mediated by the Endoplasmic Reticulum Stress Response Element in GRP78 promoter. [28] GRP78 promoter strength and luciferase gene expression increases eventually. Compared to control and other experimental setup, luciferase activity in DTT treatment setup has decreased in well 19 and 20 compared to ddH2O treatment. For other control setups, DTT treatment causes increase in luciferase activity. DTT is as reducing agent. The addition of DTT causes the unfold protein response and induce ER stress. The introduction of ER stress should cause cell apotosis and eventually cell death could occur. This shows that the increase in luciferase activity is due to random error. Also, the statistical calculations have proven that there is no significant difference between different setups. This reason cannot be justified.

Figure 13 shows the possible experimental results in the luciferase gene expression under the presence of DTT. The red arrow shows the possible reason of decrease in reading under ER stress while black arrow shows the possible reason of increase in reading under ER stress.

The second possible reason is the Genejuice causes the cytotxicity. Compare to other transfection reagent, the cytotoxicity of Genejuice transfection reagent has the minimal the cytotoxicity effect to mammalian cells. However, the Incomplete mixing of GeneJuicereagent/DNA complexes with cells could cause cytotoxicity and swirling causing the concentration effect of GeneJuice reagent/DNA complexes in center of dish. Also, it has to be noted that transfection could kill mammalian cells [].



The luminometer cannot provide accurate reading. The normalized ratio cannot be calculated accurately. Due to pipetting error, the addition of drugs may not be in correct volume for all wells.

Also, 10μL of cell lysate was used to test luciferase activity. The use of small volume could increase inaccurate reading. The dual luciferase assay can be conducted with doubling the volume of cell lysate, LAR reagent and Stop and Glo reagent.



Further Experiments

The dual luciferase assay has no significant difference in treatment of DTT and treatment with ddH2O. Experiments can be done with duplication or more. The results would be more accurate and less error.

Figure 14 shows the expected results on normalized ratio using duplication or more. (This results is obtained using group 1, group 4 and group 16 data.)

Also, the effect of DTT under different mammalian cells of gene expression is different. The effect of DTT treatment on gene expression in 293T cells should be carry out in order to understand other protective mechanisms for this type of ER stress response.

The effect of time for incubation and dosage of DTT can cause apoptotic cell death in different extent. The time interval for the incubation can be studied, for example, 24, 36 and 48 hours. [28]

The experiment can be carried by using multipipette for transferring and mixing of liquid in 24-well plate. Also, the luminometer can be used for measuring the luminescence in 24-well plate directly. This can reduce the random error for mixing of solution.



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