Acknowledgements: I would like to acknowledge and sincerely thank Dr. Jose Pinto, Dr. Diego Zorio, Dr. Adriano Martins, Dr. Igor Alabugin, Dr. Thomas Keller, Dr. Karen McGinnis, Dr. Hedi Mattoussi, Dr. Brian Miller, Omar Awan,

Matthew D’Alessandro, Jamie Johnston, David Gonzalez-Martinez and Brittany Griffin their help, guidance and support of my honors thesis project.

I would like to acknowledge and thank the founders of the Bess H. Ward research award grant.

Abstract:Quantitative polymerase chain reaction (qPCR) is an experimental technique used to determine the initial quantity of a targeted sequence in a sample, compared to a control group. The specific aim of this paper is to determine the gene expression level of various heart proteins in mice with dilated cardiomyopathy (DCM), compared to wildtype (WT) mice. It is hypothesized that various proteins will either be upregulated or downregulated, depending on their function in the myocyte. The proteins being tested are: atrial natriuretic peptide, brain natriuretic peptide, collagen I and III, triadin, S100A, sarco endoplasmic reticulum Ca2+ ATPase, phospholamban, calsequestrin, and sodium/calcium exchanger. Quantitative polymerase chain reaction experiments were performed on RNA from the left ventricle, which was extracted using RNA-bee (Tel-Test Bulletin, Catalog number CS-104B). Reverse transcription was performed using iSCRIPT Select cDNA synthesis kit (BioRad catalog number 170-8896). The solutions used for qPCR consisted of SYBR green (Quanta Biosciences, catalog number 95056-100), forward primer, reverse primer, cDNA, nuclease free water. Optimal primer concentration was determined and primer specificity was tested by blasting all primers on PubMed, and creating melting curves for each primer. The results for this paper were inconclusive as there was a problem with the mice expression their genetic mutation which resulted in DCM.

List of Tables:Table 1: Primer name, sequence and melting temperature. (Page 12)

Table 2: Blast results of all primers. (Page 15)

List of Figures:Figure 1A: Head, neck, and tail domains of one myosin protein. (Page 3)

Figure 1B: The HMM and LMM fragments. (Page 3)

Figure 1C: The S1 and S1 sub fragments of the HMM fragment. (Page 4)

Figure 2: Myosin proteins together to form a “bipolar” filament with head domains together and both ends. (Page 4)

Figure 2: Cross bridge cycle of actin and myosin in the presence of ATP. (Page 5)

Figure 3: Sarcomere contracted and relaxed. (Page 6)

Figure 5: Tm and Tn on an actin filament. (Page 6)

Figure 6: T-tubule entering into the cell. (Page 7)

Figure 7: Nanodrop reading from extracted RNA from left ventricle tissue. (Page 14)

Figure 8: Nanodrop reading from cDNA reverse transcribed from the extracted RNA in figure 7. (Page 14)

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Figure 9: Melt Curve of atrial natriuretic peptide at an initial primer concentration of 10uM. (Page 17)

Figure 10: Melt Curve of brain natriuretic peptide at an initial primer concentration of 10uM. (Page 17)

Figure 11: Melt Curve of collagen I at an initial primer concentration of 10uM. (Page 18)

Figure 12: Melt Curve of collagen III at an initial primer concentration of 10uM. (Page 18)

Figure 13: Melt Curve of Triadin at an initial primer concentration of 10uM. (Page 19)

Figure 14: Melt Curve of sarco-endoplasmic reticulum Ca2+ ATpase at an initial primer concentration of 10uM. (Page 19)

Figure 15: Melt Curve of S100A at an initial primer concentration of 10uM. (Page 20)

Figure 16: Melt Curve of calsequestrin at an initial primer concentration of 10uM. (Page 20)

Figure 17: Melt Curve of cyclophilin at an initial primer concentration of 100uM. (Page 21)

Figure 18: Melt Curve of βactin at an initial primer concentration of 10uM. (Page 21)

Figure 19: Melt Curve of Sodium Calcium Exchanger at an initial primer concentration of 10uM. (Page 22)

Figure 20: Melt Curve of phospholamban at an initial primer concentration of 10uM. (Page 22)

Figure 21A: Melt Curve of cyclophilin at an initial primer concentration of 100uM. (Page 23)

Figure 21B: Melt Curve of cyclophilin at an initial primer concentration of 10uM. (Page 23)

Figure 22A: Melt Curve of sarcoglycan α at an initial primer concentration of 100uM. (Page 24)

Figure 22B: Melt Curve of sarcoglycan α at an initial primer concentration of 10uM. (Page 24)

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Background:“The heart contracts without interruption about 3 million times a year, or a fifth of a billion times in a lifetime.” (Lodish, et al.).

Cardiac Muscle Physiology:Muscle contraction occurs when myosin and actin interact with one another. The interaction of myosin and actin is regulated by troponin and tropomyosin, which in turn is regulated by calcium (Ca 2+). (Kalyva,Parthenakis, Marketou, Kontaraki, & Vardas, 2014).

Myosin is a motor protein found in the cell. There are several different classes of myosin, however all myosin proteins share one common property, they all use the energy released in ATP hydrolysis for mechanical work in the cell. Myosin II uses this energy for muscle contraction. (Lodish, et al.)

Myosin is made up of six polypeptides, two of the polypeptides are identical and have a high molecular weight, and thus they are called “myosin heavy chains.” Each myosin heavy chain has three domains: head, neck and tail, as seen in Figure 1A. The last four polypeptides that make up myosin have a lower molecular weight and are thus called the “myosin light chains”, there are regulatory and essential light chains. (Lodish, et al.)

Myosin II can be further classified into the heavy meromyosin (HMM) fragment and the light meromyosin (LMM) fragment, as seen in Figure 1B. The HMM fragment consists of the S1 and S2 sub fragments, as seen in Figure 1C. The head domain consists of the actin binding sites, which allow myosin-actin interactions, as well as the ATPase activity. The neck domain consists of the myosin light chains, both regulatory and essential, and the tail domain consists of the two myosin heavy chains in a coil-coil interaction. (Lodish, et al.)

Figure 1B: The HMM and LMM fragments. Source: (Lodish, et al.), labels edited for paper.

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Figure 4A: Head, neck, and tail domains of one myosin protein. Source: (Lodish, et al.), labels edited for paper.

Figure 1C: The S1 and S1 sub fragments of the HMM fragment. Source: (Lodish, et al.), labels edited for paper.

As seen in Figure 2, many different myosin’s cluster together to form a bipolar filament.

Figure 2: Myosin proteins together to form a “bipolar” filament with head domains together and both ends. Source: http://www.pha.jhu.edu/~ghzheng/old/webct/note2_3.htm

Actin assembles into a polymer, F-actin that is made up of many linear G-actin monomers, can be seen in Figure 3 below. Actin has functionally distinct ends, (+) end and (-) end. The difference in the ends of actin is that actin monomers favor the addition to the (+) end over the (-) end. (Lodish, et al.)

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Cross-Bridge Cycle:

When no ATP is present, the myosin head is tightly bound to actin. When one ATP molecule binds to the myosin head domain, the myosin loses affinity for actin, thus myosin and actin are no longer bound. The myosin head then hydrolyzes the ATP to ADP and Pi, which rotates the myosin head into a “cocked state.” In this cocked state, the myosin head stores the energy released during ATP hydrolysis. Also in this cocked state, the myosin head binds to actin. Upon the release of ADP and P i

the stored energy is also released, resulting in a “power stroke” which essentially moves the myosin head along the actin. The exponential repetition of this cycle generates muscle contraction. This interaction that occurs can also be known as a “cross-bridge cycle”, shown in Figure 3. (Lodish, et al.)

Figure 5: Cross bridge cycle of actin and myosin in the presence of ATP. Source: http://chemistry.elmhurst.edu/vchembook/615coricycle.html

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In each cardiac muscle cell, there are many myofibrils that are composed of a repeating array of sarcomeres, which are the basic unit of contraction. Each sarcomere is made up of the thick and thin filaments, as shown in Figure 4. The thick filaments are composed of the myosin II bipolar filaments. The thin filaments are composed of the actin filaments which are assembled with their (+) ends orientated in the same direction, towards the Z disk. During

the cross-bridge cycle, the myosin heads move toward the Z-disk and essentially shorten the sarcomere and thus the muscle contracts. (Lodish, et al.)

Figure 6: Sarcomere contracted and relaxed. Source: https://en.wikipedia.org/wiki/Myosin.

Actin and myosin are not always able to interact with one another. Their interaction is regulated by the tropomyosin (Tm) and troponin (Tn) complex. (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas,2014). When Tm is bound to actin, it blocks the myosin binding sites, thus inhibiting muscle contraction, as seen in Figure 5. (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014). The Tn protein is made up of three subunits: Troponin I (TnI), Troponin C, (TnC), and troponin T, (TnT). (Willott, et al., 2009)

Each subunit plays a specific role in muscle contraction and relaxation. Troponin I is an ATPase inhibitory protein, TnC is a Ca2+ binding protein, and TnT binds the entire Tn complex to Tm. (Willott, et

al., 2009) The Tm and Tn inhibition can be removed in the presence of calcium (Ca2+). (Lodish, et al.)

Figure 5: Tm and Tn on an actin filament. Source: https://www.studyblue.com/notes/note/n/chapter-10-continued/deck/1342618.

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The C- and N- domains of TnC both consist of EF hand motifs. (Kalyva, Parthenakis, Marketou, Kontaraki,& Vardas, 2014). The C-domain has two binding pockets for a cation, while the N-domain has only one binding pocket. It has been shown that the C-domain binding sites are always occupied with either Ca 2+

or Mg2+, depending on the concentration of each cation in the cytosol. (Kalyva, Parthenakis, Marketou,Kontaraki, & Vardas, 2014). Under the resting concentration of 1mM Mg2+, the N-domain binding site has been shown to have a 33-44% saturation with Mg2+. (Kalyva, Parthenakis, Marketou, Kontaraki, &Vardas, 2014)

However, when the concentration of Ca2+ in the cytosol is increased past its resting concentration of below 0.1μM (Lodish, et al.) to 10μM during an action potential, (Kalyva, Parthenakis, Marketou,Kontaraki, & Vardas, 2014) Ca2+ binds to the N-domain binding site, which in turn causes a conformational change of TnI and Tm, which releases the Tm actin-myosin inhibition and allows myosin to bind to actin. (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014). Also, the Ca2+ binding to TnC relieves the TnI inhibition of ATPase activity. (Willott, et al., 2009) Therefore, muscle contraction occurs.

Excitation-Contraction Coupling: During an action potential, Ca2+ travels from the sarcoplasmic reticulum (SR), where it is bound to calsequestrin (CSQ) (Liew & Dzau, 2004) to the myocyte cytosol via proteins called ryanodine receptors (RyR). (Zima & Terentyev, 2013) The RyR channels only release the SR Ca2+ when they are activated,

which happens as a result of a small Ca2+ current via the L-type channels on the T-tubules on the sarcolemma, as shown in Figure 6. (Zima & Terentyev, 2013)

Thus, the Ca2+ release from the RyR channels is referred to as Ca2+ induced Ca2+ release. (Zima & Terentyev, 2013). Once in the cytosol, the Ca2+ binds to TnC, and muscle contraction occurs. Muscle relaxation occurs when the Ca2+ is removed from the cytosol. This Ca2+ removal can happen via the protein sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2A) which transports Ca2+ back into the SR, or the Ca2+ can exit the cell via plasma membrane calcium ATPase (PMCA) or the sodium/calcium exchanger (NCX). (Liew & Dzau, 2004). SERCA2A is regulated by the protein phospholamban (PLB). If PLB is phosphorylated by protein kinase A (PKA), it will

disassociate from SERCA2A, and thus the inhibition is removed and SERCA2A can reuptake CA2+ into the SR. (Liew & Dzau, 2004).

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Figure 6: T-tubule entering into the cell. Source: (Zima & Terentyev, 2013)

Molecular Markers for Heart Disease:The protein atrial natriuretic peptide (ANP) functions to maintain cardiac homeostasis. This vasodilator protein is released in the heart ventricle in response to high blood volume and cardiac hypertrophy. A similar protein, brain natriuretic peptide (BNP) has also been shown to have an increased synthesis during cardiac hypertrophy (Gardner, 2003). Other proteins that has been shown to have an altered expression level during heart disease are Collagen Type I and III (Col I and Col III, respectively) (Pauschinger, Knopf, Petschauer, & Doerner, 1999). Collagen fibers can be described as proteins that provide “structural integrity, mechanical strength, and resilience.” (Lodish, et al.) This altered expression level may mean many things for the heart muscle, as each type of Collagen has its own properties. For example, Collagen I is mainly in stiff tissues, while Collagen III is mainly found in more elastic tissues. Another important protein is S100A, which is a member of the S100 protein family. S100A is a Ca2+ binding protein that interacts with SERCA2A and PLB, and it has been shown that an increase in S100A1 results in an increase in SERCA2A activity. (Duarte-Costa, Castro-Ferreira, Neves, & Leite-Moreira, 2014). One final important protein that may have a change in expression during heart disease is the protein triadin (trdn). Triadin (trdn) functions to connect RyR to the Ca 2+ buffering protein, CSQ. (Chopra & Knollmann, 2013).

Introduction:The heart disease dilated cardiomyopathy (DCM) is a disease that affects the contraction of the heart muscle. This disease often results in an enlargement or dilation of the left ventricle, hence the name dilated cardiomyopathy. (Hershberger, Hedges, & Morales, 2013). During this disease state, the heart will try to maintain asymptomatic and perform normal cardiac functions (Liew & Dzau, 2004). This may result in an altered expression level of certain proteins in the heart, compared to a control wildtype (WT) heart that does not have a mutation.

Specific Aim:The specific aim of this paper is to determine the gene expression level of various heart proteins in mice with a DCM disease, compared to WT mice. To quantify the levels of gene expression, quantitative polymerase chain reaction (qPCR) was used. The hypothesis to be tested is that the diseased heart will have an upregulation or downregulation of various heart proteins in response to the DCM disease.

Quantitative polymerase chain reaction: Quantitative polymerase chain reaction (qPCR) is an experimental technique where specific regions of DNA or complementary DNA (cDNA, DNA synthesized from RNA) are amplified many thousand fold. The specific regions that are amplified are determined by forward and reverse primers that are added to the reaction solution. These primers are designed to bind to a specific sequence that corresponds to a specific gene, and thus only that gene sequence is amplified. (Life Technologies).

The qPCR experiments consist of three steps, which are: denaturation, annealing, and extension. (Each of these steps will be explained in detail later on in the paper). After completion of these three steps, the experiment cycles back and the cycle begins again at the annealing step for 39 cycles (LifeTechnologies).

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During the qPCR experiment, the amplified produced is measured at the end of each cycle. This is the key difference between quantitative PCR and regular PCR, as regular PCR measures the amount of amplified product produced only at the end of the reaction. Due to the product being measured at the end of each cycle, researches can use qPCR to determine the initial quantity of starting cDNA, corresponding to the region being amplified (Life Technologies).

The reaction solution includes: SYBR green (Quanta Biosciences, catalog number 95056-100), forward primer, reverse primer, cDNA, nuclease free water. SYBR green is an interlacing fluorescent dye that binds to the minor grove of any double stranded DNA, the dye only omits fluoresce when bound to a double stranded molecule. The forward and reverse primers bind to the region on the cDNA they are specific for and the nuclease free water is added to increase the volume of the solution to 10µL. (LifeTechnologies).

When the initial reaction solution is prepared, the cDNA is double stranded, thus the SYBR green dye binds to the minor groove and fluorescence occurs. During the denaturation step, the reaction is heated to 95℃. This high temperature results in the dissociation of the double stranded cDNA. Now that the cDNA is single stranded, the SYBR green will not bind to it, and the primers have room to bind and amplify. The next step in the reaction is the annealing step, the temperature of this step is important because the correct temperature is needed to allow the primers to bind to the cDNA. During the extension step, primer extension occurs at rates of up to 100 base pairs per second. The reaction then cycles back to the annealing step, and the cycle repeats 39 times. (Life Technologies).

As the extension occurs, the amplified product is double stranded, thus SYBR green binds to it. As more product is accumulated, more SYBR green is able to bind, thus more fluorescence is expressed. After each cycle, the qPCR machine measures the amount of product by measuring the amount of fluorescent signal. The baseline of the qPCR reaction is the fluorescence signal that occurs during the initial stages of the reaction. The cycle number corresponding to fluorescence that increases past the baseline threshold, is referred to as the threshold cycle (C t). The Ct is inversely related to the initial quantity of cDNA and therefore the Ct number is used for analysis. The Ct numbers generated during the reaction are analyzed by the ∆∆Ct method. This method is used for relative gene expression, aka- finding the expression of a gene relative to a control group (Life Technologies).

As previously stated, the region that is amplified depends on where the primers bind. Therefore, the accuracy of the binding results directly depends on how specific the primers are. Two ways to ensure primer specify are blasting primer sequence to ensure it matches to correct gene, and generating melting curves of primers.

Also, as previously stated the SYBR green dye binds to any double stranded molecule. Therefore, any non-specific region that is amplified or any primer-dimers that are formed will also give a fluorescent signal. A way to check that this is not happening in a reaction is to look at a melting curve of the reaction after the reaction is complete.

Melting curves are generated once the reactions 39 cycles are complete. The reaction is heated back up to 95℃, which dissociates all of the amplified product. Once the produced dissociates, it is single stranded and thus SYBR green no longer omits a fluorescent signal. The qPCR machine monitors this change in fluorescent signal and this data is what is used to generate the melting curve. If the SYBR green stops omitting a fluorescent signal at the same temperature, then the reaction amplified only one

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product. This would be shown in the melting curve as one single, sharp peak. Thus melting curves are a way to check primer specificity and a way to double check what the SYBR green is binding to. (LifeTechnologies).

As previously stated, the cDNA that is used in the qPCR reaction is synthesized from RNA. Thus the purity of RNA and cDNA also plays a role in how accurate the qPCR results are.

A way to measure RNA and cDNA purity is to use a NanoDrop spectrophotometer. The NanoDrop measures absorbance of light at different wavelengths. The ratio “260/280” refers to the ratio of absorbance at 260nm vs 280nm. A ratio of ~ 2.0 means the sample of RNA is “pure” and free of any contaminates. A lower reading may mean the sample is contaminated with phenol or ethanol solutions from the RNA extraction. This ratio is due to RNA having an absorbance at 260nm and proteins and phenol (found in the RNA-bee solution) absorb at 280nm. A ratio that is significantly below 2.0 may mean the RNA is contaminated with phenol, which could result if the extraction was not done properly (NanoDrop Technologies , 2007).

Another ratio that is used is “260/230” this ratio should be higher than the “260/280” ratio. If it is lower, this may mean there are contaminants in the sample that have a wavelength corresponding to 230nm. An example of such a contaminant would be ethanol. This could have occurred if the RNA pellet was not properly dried and all the ethanol was not removed. (NanoDrop Technologies , 2007)

An internal control gene must be used in order to normalize the data. In this paper, the internal control genes used are: β-actin, and mcyclophilin (mcyclo). It has been shown that using multiple internal control genes leads to less error, compared to using just one internal control. (Vandesompele, et al.,2002).

Materials and Methods:Mouse euthanasia and heart isolation:Six month old mice were sacrificed according to the Florida State University ACUC approved methods (approved animal protocols 1131 and 1435), hearts were quickly isolated, atria removed, and right and left ventricles separated. Ventricles were flash frozen using liquid nitrogen. Samples were stored at -80 ℃. The total time from sacrifice to flash freeze was under 30 seconds for each heart.

Heart tissue powder: All surfaces and instruments were cleansed with 70% ethanol, RNase out, and distilled water. Masks and gloves were worn at all times to ensure no sample contamination.

Left ventricle tissue was used to perform the rest of the experiments. Left ventricle was removed from -80℃ and immediately placed in liquid nitrogen to ensure no tissue thawing occurred. The left ventricle was removed from liquid nitrogen and placed into a mortar (pre-chilled with liquid nitrogen). The tissue was crushed into a fine powder using the mortar and pestle, while simultaneously pouring liquid nitrogen on sample to ensure it was still frozen. The tissue powder was then placed into a pre-chilled cryotube, which was then placed immediately into liquid nitrogen. Sample stored at -80℃ until used.

RNA extraction: All surfaces and instruments were cleansed with 70% ethanol, RNase out, and distilled water. Masks and gloves were worn at all times to ensure no sample contamination.

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RNA extraction was performed using RNA-Bee (Tel-Test Bulletin, Catalog number CS-104B) solution on the left ventricle fine powder tissue. This method is a liquid-liquid extraction. All sample centrifuging was performed at 4 ℃ and 12,000 RPM.

One mL of RNA-Bee (containing phenol) was added to about 50mg of sample. Each 1mL sample was split into two 0.5mL samples, to ensure no spillage when homogenizing. Manual pipetting to mix solution and begin the homogenization process was performed before the use of the polytron homogenizer. After homogenization, the two 0.5mL samples were added to be one 1mL sample and the solution was centrifuged for 10 minutes to gather all non-homogenized tissue in the bottom. Supernatant was removed and saved, tissue properly discarded. The samples were placed to sit on the benchtop for 5 minutes, then 0.2mL chloroform was added, the samples were vigorously shaken for 20 seconds and then placed on the benchtop for 15 minutes. Samples were then centrifuged for 15 minutes. The top aqueous layer was separated and saved without disturbing the interphase layer. To the aqueous layer, 0.5mL of isopropanol was added and samples vigorously shaken for 20 seconds and placed on benchtop for 10 minutes. Samples were centrifuged for 30 minutes. The RNA pellet is now visible in solution. Supernatant solution was carefully removed and discarded, without disturbing the RNA pellet. If the pellet was disturbed during supernatant removal, sample was centrifuged again for 10 minutes to re-create pellet. To the RNA pellet, 1mL of 75% ethanol was added to wash the pellet.

If no pellet appeared after the 30 minutes of centrifuging, the latter steps were continued still as the RNA may be present but invisible to the naked eye.

The ethanol was carefully removed without disturbing the pellet. Sample tubes were left open under the hood for 5 minutes to dry any excess ethanol. After the allotted 5 minutes, if there is a noticeable amount of ethanol still in sample, samples were dried for an extra 2 minutes. Extra precaution was taken to ensure the RNA pellet was not over dried. To the dry RNA pellet, 10uL of nuclease free water was added to retro spend the pellet. RNA quantity and purity was then measured using a NanoDrop machine. The NanoDrop was set to “nucleic acid: RNA” and the readings were performed using 1uL of sample.

This method of choice is used because RNA-bee, chloroform, and isopropyl alcohol are the best suited liquids to extract RNA. This is because phenol helps denature the tissue, chloroform helps retain water which results in a higher RNA yield, and alcohol is added to prevent foaming. (Zumbo)

Complementary DNA (cDNA) synthesis:Complementary DNA synthesis was performed using iSCRIPT Select cDNA synthesis kit (BioRad catalog number 170-8896). The desired concentration of RNA for cDNA synthesis is 1000ng/µL, therefore dilutions were performed after the NanoDrop measurements to reach a final RNA concentration of 250ng/µL, and then 4µL of this RNA solution was used. Added to the RNA was 4µL of 5X Script Reaction Mix, 1µL of reverse transcriptase and nuclease free water. The volume of water is added so the final volume of the cDNA synthesis reaction reaches 20 µL. The cDNA synthesis was performed in a polymerase chain reaction machine under the reaction conditions of 25℃ for 5 minutes, 42℃for 30 minutes, 85℃ for 5 minutes.

NanoDrop measurements were recorded again to check the quantity and purity of the cDNA. The NanoDrop was set to “nucleic acid: SS DNA”, where SS stands for single stranded. The final desired concentration of cDNA is 100 ng/µL therefore dilutions were made accordingly.

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Molecular markers to be tested:The proteins that are being tested for their gene expression are as follows: ANP, BNP, Col I, Col III, TRDN, S100A, SERCA2A, PLB, CSQ and NCX.

Primer design:Primers were designed using the Integrated DNA Technology (IDT) “Primer Quest Tool.” All mus musculus gene sequences were searched for on the “National Center for Biotechnology Information” ‘PubMed’ website, under the nucleotide section. All primers designed had a GC content between 45-55%, to ensure proper annealing.

All gene sequences used were the completed cds, meaning complete coding sequence. Once the forward and reverse primers were generated from the IDT website, they were blasted on PubMed to ensure specify.

Primers designed: Name Sequence Melting Temp*ANP F CCT AAG CCC TTG TGG TGT GT 57.4*ANP R CAG AGT GGG AGA GGC AAG AC 57.3*BNP F ACT CCT ATC CTC TGG GAA GTC 55.1*BNP R GCT GTC TCT GGG CCA TTT 55.3*COL 1 F ACG GCT GCA CGA GTC ACA C 60.6*COL1 R GGC AGG CGG GAG GTC TT 60.7*COL3 F GTT CTA GAG GAT GGC TGT ACT AAA CAC A 57.7*COL3 R TTG CCT TGC GTG TTT GAT ATT 54.2*TRDN F ACA CCA CCA AAG GCC AGA AA 57.4*TRDN R ATG GTG GTG GCA TAA CTG GG 57.6*Serca 2a F AGA TGG TCC TGG CAG ATG AC 56.7*Serca 2a R CCA GGT CTG GAG GAT TGA AC 55.1*S100A F TGG ATG AAA ACG GAG ATG GGG 57.2*S100A R TAC AAG CCA CTG TGA GAG CA 56.2*CSQ F ACG ATG GGA AAG ACC GAG TG 57.0*CSQ R GGC CAC AAG CTC CAG TAC AA 57.6*Mcyclo F TGG TGA CTT TAC ACG CCA TAA 54.2*Mcyclo R CCA TCC AGC CAT TCA GTC TT 54.9**β actin F AGT CCT GTC GCA TCC ACG AAA CTA 60.1**β actin R ACT CCT GCT TGC TGA TCC ACA TCT 60.3NCX F AGT CTC CCA CCC AAT GTT TC 54.8NCX R CTC CTG TTT CTG CCT CTG TAT 54.9PLB F TAT CAG GAG AGC CTC CAC TATT 54.5PLB R CAG ATC AGC AGC AGA CAT ATC 54.5*Sar α F CAA ATG CCT GTC TGT GAG CC 56.6*Sar α R GTG AGC GTG GTA GGT GAG TC 57.4

Table 2: Primer name, sequence and melting temperature. *Primer designed by Adriano Martins, PhD. ** Primer designed by Diego Zorio,PhD.

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Primer sequence blasting:To ensure primer specificity, all primer sequences were searched on the “Basic Local Alignment Search Tool of NCBI” (BLAST). The standard Nucleotide BLAST, “blastn” was used, the database used was “mouse genomic + transcript.”

Melting curve:The melting curve derivative results were exported from the qPCR machine. A graph of (-∆Florescence/∆T) by Temperature (℃) was created for each primer.

Optimal primer concentration:To find optimal primer concentration, two qPCR experiments were run comparing a primer concentration of 10µM to 100µM. The reaction solution contained 4uL SYBR green, 0.25 uL of each forward and reverse primer, 4.5 uL nuclease free water, and 1uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ .

qPCR protocol:The qPCR experiments were performed using the florescence molecule SYBR green (Quanta Biosciences, catalog number 95056-100). The final qPCR solution volume was 10µL and it consisted of 4µL SYBR green, 0.25µL forward primer, 0.25mL reverse primer, 2µL of 100ng/µL cDNA and 3.5 µL of nuclease free water. The volume of water can be changed to make the final solution volume to be 10µL, depending on the volume and concentration of cDNA. The concentration of each primer solution was 10µM. Primers were diluted with nuclease free water. The qPCR reaction conditions used were 4.00 minutes of 95℃ , 30 seconds of 95℃ ,30 seconds of 53℃*, 30 seconds of 72.0℃, then the reaction cycles back to the second step and repeats this for 39 cycles. After the reaction is complete, it runs for 5 seconds at 65℃ and 50 seconds at 95℃for a melting curve. (* this is the annealing step, this temperature depends on the melting temperature of the primers being used in the reaction. The temperature used in each reaction is taken as the average of all primer melting temperatures – 2).

Data analysis: The data generated during the qPCR reaction was exported onto a USB drive and transferred to an excel file where it was then analyzed during the ∆∆Ct method. This was accomplished via the following equations, as described in (Winer, Jung, Shackel, & Williams, 1999).

∆CtWT=HousekeepWT Ct−Geneof interest (GOI )WT Ct

∆CT Hetero=HousekeepHeteroCt−GOI HeteroCt

∆ ∆Ct=∆CtWT−∆Ct Hetero

2−∆∆Ct=Fold differenceof geneexpression , relative¿ the control animal

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Results:Mouse identification:

Mouse ID Whole heart weight473 0.10864 g

RNA extraction:

Figure 7: Nanodrop reading from extracted RNA from left ventricle tissue. Extraction was performed using RNA-bee, chloroform, isopropanol and 75% ethanol following the protocol listed in the materials and methods section. Sample ID used was “WT #473.”

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CDNA synthesis:

Figure 8: Nanodrop reading from cDNA reverse transcribed from the extracted RNA in figure 7. cDNA was synthesized using iScript Select cDNA synthesis kit (BioRad catalog number 170-8896) following the protocol listed in the materials and methods section. Sample ID used was “WT #473.”

Primer sequence blast:Name Sequence Blast results Query Cover Identity ANP F CCT AAG CCC TTG

TGG TGT GTMus musculus natriuretic peptide type A (Nppa), mRNA

100% 100%

ANP R CAG AGT GGG AGA GGC AAG AC

Mus musculus natriuretic peptide type A (Nppa), mRNA

100% 100%

BNP F ACT CCT ATC CTC TGG GAA GTC

Mus musculus natriuretic peptide type B (Nppb), transcript variant 1, mRNA

100% 100%

BNP R GCT GTC TCT GGG CCA TTT

Mus musculus natriuretic peptide type B (Nppb), transcript variant 1, mRNA

100% 100%

Col I F ACG GCT GCA CGA GTC ACA C

Mus musculus collagen, type I, alpha 1 (Col1a1), Mrna

100% 100%

Col I R GGC AGG CGG GAG GTC TT

Mus musculus collagen, type I, alpha 1 (Col1a1), Mrna

100% 100%

Col III F GTT CTA GAG GAT GGC TGT ACT AAA CAC A

Mus musculus collagen, type III, alpha 1 (Col3a1), mRNA

100% 100%

Col III R TTG CCT TGC GTG TTT GAT ATT

Mus musculus collagen, type III, alpha 1 (Col3a1), mRNA

100% 100%

TRDN F ACA CCA CCA AAG GCC AGA AA

Mus musculus triadin (Trdn), mRNA

100% 100%

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TRDN R ATG GTG GTG GCA TAA CTG GG

Mus musculus triadin (Trdn), mRNA

100% 100%

SERCA2A F

AGA TGG TCC TGG CAG ATG AC

Mus musculus ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (Atp2a2), transcript variant 1, mRNA

100% 100%

SERCA2A R

CCA GGT CTG GAG GAT TGA AC

Mus musculus ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (Atp2a2), transcript variant 1, mRNA

100% 100%

S100A F TGG ATG AAA ACG GAG ATG GGG

Mus musculus S100 calcium binding protein A1 (S100a1), mRNA

100% 100%

S100A R TAC AAG CCA CTG TGA GAG CA

Mus musculus S100 calcium binding protein A1 (S100a1), mRNA

100% 100%

Calseq F ACG ATG GGA AAG ACC GAG TG

100% Mus musculus calsequestrin 2 (Casq2), mRNA

100% 100%

Calseq R GGC CAC AAG CTC CAG TAC AA

Mus musculus calsequestrin 2 (Casq2), mRNA

100% 100%

Mcyclo F TGG TGA CTT TAC ACG CCA TAA

Mus musculus peptidylprolyl isomerase A (Ppia), mRNA

100% 100%

Mcyclo R CCA TCC AGC CAT TCA GTC TT

Mus musculus peptidylprolyl isomerase A (Ppia), mRNA

100% 100%

β actin F AGT CCT GTC GCA TCC ACG AAA CTA

Mus musculus actin, beta (Actb), mRNA

91% 91%

β actin R ACT CCT GCT TGC TGA TCC ACA TCT

Mus musculus actin, beta (Actb), mRNA

100% 100%

NCX F AGT CTC CCA CCC AAT GTT TC

Mus musculus solute carrier family 8 (sodium/calcium exchanger), member 1 (Slc8a1), transcript variant B, mRNA

100% 100%

NCX R CTC CTG TTT CTG CCT CTG TAT

Mus musculus solute carrier family 8 (sodium/calcium exchanger), member 1 (Slc8a1), transcript variant B, mRNA

100% 100%

PLB F TAT CAG GAG AGC CTC CAC TATT

Mus musculus phospholamban (Pln), transcript variant 1, mRNA

100% 100%

PLB R CAG ATC AGC AGC AGA CAT ATC

Mus musculus phospholamban (Pln), transcript variant 1, mRNA

100% 100%

Table 2: Blast results of all primers.

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Melting curves

Figure 9: Melt Curve of atrial natriuretic peptide at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: CCT AAG CCC TTG TGG TGT GT, reverse: CAG AGT GGG AGA GGC AAG AC.

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Figure 10: Melt Curve of brain natriuretic peptide at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃, 30 seconds at 72.0

℃, cycled for 39 times starting at step

two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95

℃ . The primer sequences used were

forward: ACT CCT ATC CTC TGG GAA GTC, reverse: ACT CCT ATC CTC TGG GAA GTC

Figure 11: Melt Curve of collagen I at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95

℃ ,30 seconds at 54℃, 30 seconds

at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: ACG GCT GCA CGA GTC ACA C, reverse: GGC AGG CGG GAG GTC TT

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Figure 12: Melt Curve of collagen III at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5

seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: GTT CTA GAG GAT GGC TGT ACT AAA CAC A, reverse: TTG CCT TGC GTG TTT GAT ATT

Figure 13: Melt Curve of Triadin at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: ACA CCA CCA AAG GCC AGA AA, reverse: ATG GTG GTG GCA TAA CTG GG

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Figure 14: Melt Curve of sarco-endoplasmic reticulum Ca2+ ATpase at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ ,

30 seconds at 95℃ ,30 seconds at 48.06℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used

were forward: AGA TGG TCC TGG CAG ATG AC, reverse: CCA GGT CTG GAG GAT TGA AC

Figure 15: Melt Curve of S100A at an initial primer

concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 48.06℃, 30 seconds at 72.0

℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: TGG ATG AAA ACG GAG ATG GGG, reverse: TAC AAG CCA CTG TGA GAG CA

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Figure 16: Melt Curve of calsequestrin at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95

℃ ,30 seconds at 48.06℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: ACG ATG GGA AAG ACC GAG TG, reverse: GGC CAC AAG CTC CAG TAC AA.

Figure 17: Melt Curve of cyclophilin at an initial primer concentration of 100uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer,

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4.5 uL nuclease free water, and 1uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: TGG TGA CTT TAC ACG CCA TAA, reverse: CCA TCC AGC CAT TCA GTC TT

Figure 18: Melt Curve of βactin at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95

℃ ,30 seconds at 56℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step

consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: AGT CCT GTC GCA TCC ACG AAA CTA, reverse: ACT CCT GCT TGC TGA TCC ACA TCT

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Figure 19: Melt Curve of Sodium Calcium Exchanger at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 53℃, 30 seconds at 72.0℃, cycled for 39

times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: AGT CTC CCA CCC AAT GTT TC, reverse: CTC CTG TTT CTG CCT CTG TAT

Figure 20: Melt Curve of phospholamban at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 3.5 uL nuclease free water, and 2uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95

℃ ,30 seconds at 53℃, 30 seconds

at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: TAT CAG GAG AGC CTC CAC TATT, reverse: CAG ATC AGC AGC AGA CAT ATC

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Optimal primer concentration:

Figure 21A: Melt Curve of cyclophilin at an initial primer concentration of 100uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 4.5 uL nuclease free water, and 1uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were

forward: TGG TGA CTT TAC ACG CCA TAA, reverse: CCA TCC AGC CAT TCA GTC TT

Figure 21B: Melt Curve of cyclophilin at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 4.5 uL nuclease free water, and 1uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃, 30

seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65

℃ and 50 seconds at 95℃ . The primer sequences used were forward: TGG TGA CTT TAC ACG CCA TAA, reverse: CCA TCC AGC CAT TCA GTC TT

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Figure 22A: Melt Curve of sarcoglycan α at an initial primer concentration of 100uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 4.5 uL nuclease free water,

and 1uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54

℃, 30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at 65℃ and 50 seconds at 95℃ . The primer sequences used were forward: CAA ATG CCT GTC TGT GAG CC , reverse: GTG AGC GTG GTA GGT GAG TC

Figure 22B: Melt Curve of sarcoglycan α at an initial primer concentration of 10uM. The reaction solution contained 4uL SYBR green (Quanta Biosciences, catalog number 95056-100), 0.25 uL of each forward and reverse primer, 4.5 uL nuclease free water, and 1uL cDNA. The reaction conditions used were 4.00 minutes at 95℃ , 30 seconds at 95℃ ,30 seconds at 54℃,

30 seconds at 72.0℃, cycled for 39 times starting at step two. The melting curve step consisted of 5 seconds at

25

65℃ and 50 seconds at 95℃ . The primer sequences used were forward: CAA ATG CCT GTC TGT GAG CC , reverse: GTG AGC GTG GTA GGT GAG TC

Discussion:To ensure primer specificity, all primer sequences were searched on the “Basic Local Alignment Search Tool of NCBI” (BLAST). The standard Nucleotide BLAST, “blastn” was used, the database used was “mouse genomic + transcript.” The results show that each primer had at least a 90% match to the query and identity. Meaning that the identity of the primer matches to the sequence of mouse DNA (query). This is shown in table 2.

The melting curves in figures 9-20 show the final way that primer specificity was tested. The primers in these figures all show one peak, occurring at the same temperature. This one peak is showing that amplicon product is dissociating at the same temperature, as that is the temperature that the SYBR green stops omitting a fluorescence single. Figures 21-22 show melting curves from different primer concentrations. These melting curves were used to find the optimal primer concentration. Figure 22A and 22B show a melting curve that does not contain just a single peak. However, when comparing figures A and B together, it is shown that in figure B, there are more single dissociation peaks than in A. This is due to 10µM being the optimal primer concentration to use. The concentration of 100 µM is too high, and the excess primes may form primer-dimers (two primers binding to themselves). (LifeTechnologies). Since a primer-dimer is double stranded, SYBR green binds and omits a fluorescence signal. However, the primer-dimer will not have the same melting temperate as the amplicon product, thus resulting in multiple dissociation peaks. To ensure that differences in qPCR machine readings did not interfere with the data, both 10uM and 100uM concentrations were run on the same plate

The NanoDrop measurements show that the RNA extraction and cDNA synthesis were successful as the 260/280 and 260/230 ratio readings were in the correct range.

Data analysis to determine the difference in gene expression was not performed because the mice model used in the lab was not able to correctly express the mutation that resulted in DCM.

Conclusion: The hypothesis was not able to be tested due to the mice model not being ready on time in the lab. However, this paper describes how to successfully measure the gene expression content in a tissue sample.

The primers were blasts on PubMed with the goal of determining the correct binding location. The optimal initial primer concentration was determined by running multiple plates with two of the most commonly used primer concentrations. The melting curves were performed with the goal of locating a single dissociation peak.

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The NanoDrop measurements will ensure the researched that their RNA extraction and cDNA was successful and therefore, the researcher will be able to successfully quantity gene expression.

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