experiments in this paper done through instruction in graduate course: Biotechnology laboratory in the natural sciences and mathematics department at the university of texas at dallas, RICHARDSON, TX 75080 march 2016
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γ-globin analysis by expression profiling through RT-qPCR, quantification through ELISA, and oxidative stress management analysis by FACS from KU812F cells under treatment by δ aminolevulinic acid, succinylacetone, and N-methyl mesoporphyrin
Shaan Sarode, Jose Cordero, and Dr. Li Liu experiments in this paper done through instruction in graduate course: Biotechnology laboratory in the natural sciences and mathematics department at the university of texas at dallas, RICHARDSON, TX 75080 march 2016
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γ-globin analysis by expression profiling through RT-qPCR, quantification through ELISA, and oxidative stress management analysis by FACS from KU812F cells under treatment by δ aminolevulinic acid, succinylacetone, and N-methyl mesoporphyrin
Shaan Sarode, Jose Cordero, and Dr. Li Liu

ABSTRACT
Hemoglobinopathies refer to a group of blood related disorders that encompass important disease such as thalassemia and sickle cell disease. Because many of these disease are hereditary more aggressive genetic therapies are showing promise as possible avenues of treatment. One such method is to re-express fetal hemoglobin (HbF) in hopes that it will take over the role as main functional hemoglobin.
In this paper we aim to build upon previously utilized proteomic approaches to study γ-globin by using techniques to test for direct gene expression of whole hemoglobin rather than fragmented subunits of the multimer. We hope to achieve this through studying intracellular RNA levels and transcription by RT-qPCR and hemoglobin detection methods such as enzyme linked immunosorbent assay (ELISA). We also conducted expression profiling through measuring protein expression levels of γ-globin with treatment of various chemicals including δ-aminolevulinic acid (ALA) and N-methyl mesoporphyrin (NMMP) with ELISA. It has already been established that ALA has ability to induce expression of γ-globin in adult cells but these set of experiments will use a new cell line called KU812F and new treatment of NMMP that has not been done before. We were able to show that these KU812F cells were able to produce an upregulation of γ-globin transcript globally when treated by SA more so than treated with ALA or in combination of the two. This may go to show possible benefit in this reagent being used for future genetic therapies.

INTRODUCTION
Biotechnology industry has experienced exponential growth over the past half-century and with it medicine has been able to provide newer and exciting therapies. Humans have to come to depend on introduction of novel techniques and approaches to being able to combat whatever future life challenges us with. While most of these conditions will takes years to study some are experiencing major headway into developed approaches to form cures or inspire development of another avenue of research.
Hemoglobinopathies are a group of disease characterized by deregulation or loss of function of hemoglobin protein which is an abundant protein necessary for some vital biological processes. Recently there has been a push to study different hemoglobin protein subunits. Hemoglobin exists is multiple forms consisting of differing subunits of the globin peptides. Adult hemoglobin, for example, contains two subunits of α-globin and two subunits of β-globin. In contrast fetal hemoglobin consists of two subunits of α-globin and two subunits of γ-globin. A common example of a hemoglobinopathy is sickle cell disease characterized by mutations in globin peptide genetic sequence resulting in conglomeration of hemoglobin proteins inside a red blood cells causing sickle-shaped red blood cells with symptoms of anemia, long term organ damage and eventually organ failure 1. Because many of these disease are hereditary meaning genetic information passed down from one generation to another, it is difficult to make lifestyle changes to combat these illness. Recent survey data approximates around 1.5% screened infants have some sort of hemoglobinopathy 2. Therefore it has become imperative to intervene with gene therapies such as possibly trying to re-express fetal hemoglobin (HbF), a form of hemoglobin involved in fetal oxygen transport, in adults. This would effectively replace an affected adult individual’s defunct hemoglobin with another form of hemoglobin which can be more efficient for vital biological processes 3.
In this paper we set out to examine the possibility of inducing expression of γ-globin in KU812F cells by treatment of certain chemicals. One of the chemicals used, ALA, is a known main regulatory compound in the heme synthesis pathway; Heme will form the center functional unit of the eventual hemoglobin molecule 4. We hypothesize that with induction by treatment of KU812F cells with δ-aminolevulinic acid we should see an increase of γ-globin transcripts due to increased expression. After this we will test whole protein identification by enzyme linked immunosorbent assay (ELISA) of KU812F cells having undergone different treatment which should result in increased protein amount in ALA treated cell cultures. Lastly we utilized the same treatment of KU812F cells as before with ALA or NMMP and performed fluorescence activated cell sorting (FACS). We had placed these cells under oxidative stress in hopes in being able to determine the viability of these cells under stress which we hypothesized would be very high due their function in transporting and containing oxygen derived from function of γ-globin proteins.
Overall we believe that this data will aid in the future use of ALA and other γ-globin inducing reagents in the therapy of individuals with hemoglobinopathies. With this data pertaining to oxidative stress one can conclude that reactive oxygen species may not be as a big of detriment in γ-globin expression in cells as once thought.
MATERIALS AND METHODS
RNA extraction
Cells grown in mono layer are lysed directly in a culture dish by adding the RNA STAT-60TM
(1 ml/3.5cm petri dish) and passing the cell lysate several times through a pipette. Next 0.2 mL chloroform added for every 1 mL of RNA STAT solution added, stored 2-3 minutes and centrifuged at 12000g for 15 minutes at 4o C. Solution should appear to have lower red phenol phase and clear aqueous phase which was removed and 0.5 mL isopropanol added for every 1 mL RNA STAT solution used. Samples were centrifuged after being briefly stored at 12000g for 10 minutes at 4o C. RNA precipitated and appeared to be a white pellet at this point. Supernatant with extraneous debris was removed and 200µL 70% ethanol added and centrifuged at 7500g for 5 minutes at 4o C. Ethanol was removed and RNA containing pellet was allowed to dry for a brief period. RNA pellet was suspended in RNase-free solutions for better results. 5 Small amount of RNA extraction solution was subjected to purity test to determine 260/280 ratio. Results can be seen in Table 1. The RNA samples were also loaded onto a 1% agarose gel for analysis of integrity and quality which can be seen in Figure 1.

Reverse Transcription

Previous RNA samples were obtained that were inside set purity threshold from KU812F cells. Reverse transcription reactions were set-up using standard protocol. Primer added to RNA in ratio of 1:4 respectively in 3 different sets: control (untreated), 1mM succinylacetone (SA) treated, 1mM aminolevulinic acid (ALA), or treated with 1mM both SA and ALA each of which was repeated in triplicate. The RNA samples were then incubated at 70o C for 5 mins then at 4o C for 10 mins. Master mix solutions were set up according to Figure 2.

Figure 2: Master mix solutions containing total volume of 15µL of each of the reagents listed to give final concentration in second column. Each treatment was done in triplicate
Figure 2: Master mix solutions containing total volume of 15µL of each of the reagents listed to give final concentration in second column. Each treatment was done in triplicate

To start the revers transcription process final reaction sets were made. Each reaction contained 5µL isolated RNA and 15µL of reverse transcription mixture as shown in Figure 2. BioRad thermocycler was used with program containing the following steps. First incubation at 25°C for 5 min, second incubation at 42°C for 60 min, and final incubation at 70°C for 15 min. At this point it is expected to have working solutions containing whole cell cDNA for use in quantitative polymerase chain reaction (qPCR). Samples were stored at -20°C for until used in qPCR.

RT qPCR

Previous cDNA were utilized for RT qPCR. PCR reactions involved the 274/276 set of primers for γ-globin detection and primers 291/292 for Glyceraldehyde 3-Phophate dehydrogenase (GAPDH). Primer set with sequence as shown in Figure 3. 137.5µL of nuclease-free water added to each reaction of γ-globin and GAPDH along with 312.5µL JumpStart Sybr Green and 25µL of each corresponding forward and reverse primers. For standard curve development serial dilutions of γ-globin and GAPDH were performed beforehand. 5µL of cDNA was used for each reaction. Samples were loaded in 96-well plate and into BioRad aPCR machine following the protocol listed below.
Cycle 1 (1X) at 95.0ºC for 1:30, Cycle 2 (40X, 3 steps each time) Step 1: 95.0ºC for 00:30, Step 2: 56.0ºC for 00:45, Step 3: 72.0ºC for 01:00, data collection enabled at this point. Cycle 3(1X) at 12.0ºC. Upon completion of PCR reaction cycling samples were held in this state.

Primer sets for detecting γ-globin gene:

Figure 3: Primers used for γ-globin detection and GAPDH detection listed in 5’ to 3’ direction
Figure 3: Primers used for γ-globin detection and GAPDH detection listed in 5’ to 3’ direction 274: 5' GGC AAC CTG TCC TCT GCC TC 3' 276: 5' GAA ATG GAT TGC CAA AAC GG 3'

Primer set for detecting GAPDH gene:

291: 5' GAA GGT GAA GGT CGG AGT 3' 292: 5' GAA GAT GGT GAT GGG ATT TC 3'

Enzyme linked Immunosorbent assay (ELISA)
Next to measure effect of different treatments of KU812F cells by various chemicals ELISA was performed to determine intracellular concentrations of fetal hemoglobin. ELISA protocol mainly followed from protocol given by Bethyl Laboratories, Inc. where reagents for ELISA were obtained, CAT#E101.
A sandwich method of ELISA was used for this experiment therefore 45 well plate was first coated with capture antibody which was Human HbF antibody from Cat# A80-136A, Bethyl Laboratories, Inc, incubated for 1 hour and washed several times after. Blocking solution (50mM TRIS, 0.14M NaCl, 1% BSA at pH 8.0) was added, plate incubated for 30 minutes and washed several times. Hemoglobin calibrator stock solution obtained from Cat#RC80-135-5, Bethyl Laboratories Inc. was diluted 1:2 giving final concentrations of 400ng/ml, 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, and 6.25 ng/ml.
Samples were prepared from KU812F cells treated by either nothing, N-methyl mesoporphyrin (NMMP), aminolevulinic acid (ALA) or both. Culture were lysed and collected and diluted in sample diluent (50mM TRIS, 0.14 NaCl, 1% BSA, 0.05% Tween 20) for total of 1:4 dilution of protein lysate. Samples were loaded into plate in triplicate and each sample of the triplicate done in duplicate. Plate was incubated for 1 hour and washed 5 times. Secondary antibody was conjugated to enzyme and therefore enzyme substrate (Tetramethylbenzidine) was added next, reaction was done in dark due to photosensitivity of horse radish peroxidase enzyme. Plate was incubated for 5-15 minutes or more precisely until colorimetric change was observed and reaction as stopped using 2M H2SO4 to stop the reaction. Lastly absorbance at 450nm was measured as shown in Figure 5.
Fluorescence activated cell sorting (FACS)
KU812F cells were treated similar to previous treatments used in ELISA analysis with either no treatment, ALA, NMMP or treated with both ALA and NMMP. H2DCFDA was prepared in warm sterile PBS and incubated for one hour before harvesting for FACS. Cells were centrifuged at 1000rpm for 3 minutes and added washed with PBS, this procedure was repeated two more times. Cells were then transferred to specialized FACS tubes obtained from BD falcon, Ref#352024) for FACS analysis. Cells were detected on FTIC channel (green fluorescence) on FACS Calibur and KU812F cells not treated with H2DCFDA was used as a negative control giving a background threshold value.
RESULTS

This series of experiment first set out to observe differences in gene expression through the studying of RNA transcript formation in KU812F cells of γ-globin.
Table 1: Data obtained from nanodrop spectrophotometer showing RNA concentration per µL and absorbance at 260 and 280 nm
Table 1: Data obtained from nanodrop spectrophotometer showing RNA concentration per µL and absorbance at 260 and 280 nm
The first method done was to extract RNA transcripts of the entire cell at a certain time point from KU812F cells to note the baseline amount of γ-globin transcripts are present in an non-induced cell. Table 1 shows the results obtained from running 1µL of obtained RNA sample on BioRad nanodrop spectrophotometer (courtesy of Winkler Lab). Nucleic Acid Conc. | Unit | A260 | A280 | 260/280 | 260/230 | Sample Type | 2064.7 | ng/µl | 51.616 | 25.742 | 2.01 | 1.58 | RNA |

Once spectrophotometer data was obtained, RNA was subjected to gel electrophoresis as shown in Figure 1. This was to test for purity of rRNA within the extracted RNA sample.

Figure 1: Shows extracted RNA run on 1% agarose gel at 150 V for 30 minutes. Bands indicated as labelled. Multiple lanes were run with lower quality RNA
Figure 1: Shows extracted RNA run on 1% agarose gel at 150 V for 30 minutes. Bands indicated as labelled. Multiple lanes were run with lower quality RNA
Degraded RNA
Degraded RNA
18S RNA
18S RNA
28S RNA
28S RNA

Next in the protocol was to perform reverse transcription on the extracted RNA. After these cDNA fragments were manufactured we aimed to process and amplify them through the qPCR process. This would allow us to quantify and compare relative transcript amounts between various samples. The program also was enabled to provide raw data results which were used in quantifying relative fold change (FC) with associated standard deviations as shown in Table 2. Bar graph showing relative fold change with standard deviations is as shown in Figure 4. Table 2: Data analysis of raw data provided by iCycle PCR program showing effect of treatment by either ALA or SA on expression of γ-globin

Figure 4: Histogram showing relative fold change when comparing various treatments of KU812F cells measuring expression of γ-globin gene. The values used to compare fold changes are the γ globin-GAPDH ratio. GAPDH used as a reference gene as its expression is unchanged with treatments used in this experiment. RNA amount used for each reaction was 5µL of 2064 ng/µL RNA. Error bars here represent the standard deviation of each set of treatments which were done in triplicate.
Figure 4: Histogram showing relative fold change when comparing various treatments of KU812F cells measuring expression of γ-globin gene. The values used to compare fold changes are the γ globin-GAPDH ratio. GAPDH used as a reference gene as its expression is unchanged with treatments used in this experiment. RNA amount used for each reaction was 5µL of 2064 ng/µL RNA. Error bars here represent the standard deviation of each set of treatments which were done in triplicate.

Table 5: Spectrophotometer data of absorbance at 450nm of standards and HbF samples. Column 1 and 2 are standards duplicated. A3-F3 are triplicated duplicates of control untreated cells, G3-D4 are triplicated duplicates of 1mM SA treated KU812F cells, E4-B5 are triplicated duplicates of 1mM ALA treated KU812F cells, and D5-H5 are triplicated duplicates of 1mM SA + 1mM ALA treated KU812F cells
Table 5: Spectrophotometer data of absorbance at 450nm of standards and HbF samples. Column 1 and 2 are standards duplicated. A3-F3 are triplicated duplicates of control untreated cells, G3-D4 are triplicated duplicates of 1mM SA treated KU812F cells, E4-B5 are triplicated duplicates of 1mM ALA treated KU812F cells, and D5-H5 are triplicated duplicates of 1mM SA + 1mM ALA treated KU812F cells
The next part of the experiment series consisted of analysis and quantification of HbF through detection of γ globin subunit by ELISA. Table 5 shows spectrophotometer readings and Figure 5 shows corresponding standard curve for protein concentration determination. | 1 | 2 | 3 | 4 | 5 | A | 0.064 | 0.089 | 0.595 | 0.232 | 1.598 | B | 1.84 | 1.787 | 0.537 | 0.342 | 1.543 | C | 1.721 | 1.836 | 0.721 | 0.271 | 0.239 | D | 1.447 | 1.372 | 0.607 | 0.32 | 0.347 | E | 0.787 | 0.778 | 0.567 | 1.404 | 0.245 | F | 0.404 | 0.39 | 0.479 | 1.33 | 0.225 | G | 0.232 | 0.202 | 0.245 | 1.261 | 0.498 | H | 0.128 | 0.135 | 0.291 | 1.267 | 0.28 |

Figure 5: Standard curve with standard errors bars showing Bradford assay data as a curve for use in protein concentration determination. Calibrator stock Hb was used obtained from Bethyl Laboratories and serial dilution was performed to obtain concentrations.
Figure 5: Standard curve with standard errors bars showing Bradford assay data as a curve for use in protein concentration determination. Calibrator stock Hb was used obtained from Bethyl Laboratories and serial dilution was performed to obtain concentrations.

The final experiment of this series was the FACS analysis which was to obtain information about reactive oxygen species inside the intracellular environment of KU812F cells. This was done through FACS analysis. Table 6 shows data analysis of raw data obtained from FACS machine showing fold change and standard deviation relative for treatments types. Treatment | Replicate | % fluorescent cells | Mean | Std. Dev. | Fold change % of fluorescent cells | Std. Dev. | control | 1 | 2.76 | 2.89 | 0.169901932 | 1 | 0.058789596 | | 2 | 2.78 | | | | | | 3 | 3.13 | | | | | NMMP | 4 | 70.11 | 68.09 | 1.736740242 | 23.56055363 | 0.60094818 | | 5 | 65.87 | | | | | | 6 | 68.29 | | | | | ALA | 7 | 82.6 | 83.38 | 0.575210107 | 28.85121107 | 0.199034639 | | 8 | 83.97 | | | | | | 9 | 83.57 | | | | | NMMP + ALA | 10 | 77.34 | 76.22666667 | 1.674461771 | 26.37600923 | 0.579398537 | | 11 | 77.48 | | | | | | 12 | 73.86 | | | | |

Table 6: Raw data and analysis for FACS, with various treatments of KU812F cells be either ALA, NMMP or both (1mM). Fold changes are calculated as in reference to control.
Table 6: Raw data and analysis for FACS, with various treatments of KU812F cells be either ALA, NMMP or both (1mM). Fold changes are calculated as in reference to control.

DISCUSSION

In our first experiment of RNA extraction it can be seen that our RNA is within the standard region of 1.8-2.0 in respect to 260/280 ratio. This was originally established by J. Glasel in 1995 and has been used as a standard for RNA purity examination 6. As the data shows from the nanodrop spectrophotometer that the extracted RNA purity is very high at over 2.0. This may also be confirmed by gel electrophoresis showing low amount of RNA degradation products (the last band) and high thick amount of rRNA.
The qPCR process was used to amplify cDNA fragments for detection and comparison of different treatment styles of cell cultures these original cDNAs were obtained from. Our results in Figure 4 show most fold change increase in cell treated by succinylacetone (SA). This result however is counter-intuitive as SA is a natural inhibitor of ALA dehydrogenase, an enzyme responsible for heme synthesis. This may prove that there is an alternate path that built up ALA which has not been oxidized yet can still feed into the heme synthesis pathway. However when we treated cells with both SA and ALA, we saw a smaller than just SA but larger than untreated gross fold change increase which may confirm the previous hypothesis of an alternate pathway for ALA to become heme.
Lastly we used ELISA to measure HbF detection through use of antibodies. We saw that the cells treated with ALA had the most detectable HbF. However this is in contrast with our previous result. We may be able to explain this result by understanding the phenomenon of sandwich method used in our ELISA protocol. Because we used an antibody specific to γ-globin, it may be that this epitope existed in denatured HbF and therefore may produce a false positive result whereas the reality may have been that this HbF was indeed non-functional but still containing the epitope of γ-globin.
In our final experiment we used FACS analysis to be able to study oxidative stress on KU812F cells. This would be worth due to known increased affinity for oxygen in HbF, therefore if possible future genetic therapies proved to point towards increasing HbF concentration in adult cells we would like to study the effect of that extra oxygen in an intracellular environment. We used hydrogen peroxide due to its importance in cell cycle regulation. H2DCFDA is non-fluorescent but when trapped inside the cell and cleaved and oxidized by hydrogen peroxide it becomes fluorescent as DCF (dichlorofluorescein) which can be detected at 522nm. Our FACS analysis results show that there is an increase of fluorescence detection in cells in ALA treated cells which is consistent with our previous hypothesis or ALA being an inducer of γ-globin in KU812F cells. This result is important because it may show that these cells are most capable to handle reactive oxygen species and therefore this reagent combined with these cells may be ideal for future genetic therapies of individuals with hemoglobin deficiencies.
This series of experiment set out to test whether or not fetal hemoglobin can be induced to be expressed in adult cells. We have proven here the by treatment with ALA and NMMP we can increase γ-globin expression by at least 1 fold and therefore have proved our hypothesis. If this was the case then we may see future application of these results in being able to provide gene therapies for individuals suffering from hemoglobinopathies.
REFERENCES

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