










































  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

 
 

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. 

CHARACTERIZING AN  
ALTERNATIVELY SPLICED 

VARIANT OF  
CHEMOKINE RECEPTOR 2 

IN PAINFUL DIABETIC 
NEUROPATHY 

JUSTINE SOLTYS 
LEI YU (FACULTY ADVISOR) 

 

 

✵ ABSTRACT 
Prior research efforts have demonstrated a 

link between neuroinflammation and the progres-
sion of Painful Diabetic Neuropathy (PDN), a chronic 
cascade of nerve damage that presents as tingling, 
numbness, hypersensitivity to touch, or intense pain. 
Current treatments are focused on pain manage-
ment, serving to temporarily mask these symptoms 
without repressing or slowing nerve damage. The 
chemokine-receptor system has been closely stud-
ied for its role in perpetuating neuropathic pain, alt-
hough its precise mechanistic involvement remains 
unclear due to the network’s complexity. Because of 
its likely role in regulating neuropathic pain, target-
ing CCR2 may be the key to effective treatment of 
PDN.  

Alternative splicing of CCR2 leads to two dis-
tinct isoforms with different C-terminus sequences, 
CCR2A and CCR2B. The present study was intended 
to differentiate between these isoforms through spe-
cific primer design, selection of optimized pairs, RT-
PCR, and amplicon sequencing to verify the PCR 
products. However, the study has revealed a third, 
previously unreported isoform, CCR2C, due to evi-
dence of alternative splicing and both the absence 
and insertion of parts of A and B. In the long term, 
we predict that the relationship between CCR2’s al- 

ternatively spliced transcript variants will lead to a 
distinct pattern of isoform prevalence in individuals 
suffering from PDN. Discerning the genetic profiles 
of patients with PDN and healthy individuals will clar-
ify the complex mechanism driving CCR2’s intracel-
lular interactions and offer more effective therapeu-
tic options.    
 

1 INTRODUCTION 
PAINFUL DIABETIC NEUROPATHY 

Painful Diabetic Neuropathy (PDN) gives rise 
to several types of peripheral nerve damage but 
most frequently affects the feet in a pattern of distal-
to-proximal severity. During disease progression, 
the extremities are more severely impacted than the 
midline of the body, which models the branching 
peripheral nervous system. Those neurons com-
prised of the longest axons exhibit increased expo-
sure due to their size and distribution, thus sustain-
ing the brunt of the damage.[11] As the sensory 
nerves are affected, individuals may experience 
numbness tingling, or a loss of reactivity to sensa-
tions like temperature, pain, or touch. Damage to 
the motor nerves causes hyperalgesia (enhanced 
pain sensitivity) and allodynia (hypersensitivity to 
neutral stimuli often felt as a stabbing pain).[20] The 
incidence of PDN within diabetes ranges between 
10–26%, reflecting differences among sample pop-
ulations.[2] 

In individuals with PDN, a number of related 
metabolic and vascular factors are implicated in the 
onset of nerve damage. Nerve damage stimulates 
the infiltration of macrophages, leading to an in-
crease in the number of pro-inflammatory cytokines 
circling throughout the body. Clinical studies involv-
ing diabetic neuropathic (DN) subjects with and 
without pain demonstrate that groups experiencing 
PDN have higher inflammation markers and in-
creased cytokine concentration. The neuroinflam-
matory and immune responses are thus heightened 
and contribute to the development of neuropathic 
pain.[1] 
 Several factors are believed to contribute to 
the pain mechanism of PDN; as a result, current 
treatment options are decidedly limited. Glycemic 



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

control, anticonvulsants, antidepressants, and opi-
ates are meant to assuage pain and other symptoms 
related to PDN, but fail to restore nerve function.[13] 
Tightened glycemic control lowers the blood sugar 
levels in an effort to reduce the risk of hyperglyce-
mia, a causative factor in neuropathy. While this 
practice has displayed some success in slowing the 
progression of PDN, it does not alleviate nerve pain, 
and other drugs vary in the consistency of their re-
sults.[6] Individuals suffering from PDN continue to 
live with nerve pain, loss of sensation, and the risk of 
limb amputation. Because of this, there is still a 
pressing need to understand how PDN manifests 
and to develop effective therapeutic options that 
treat PDN directly.  
 
THE ROLE OF CCR2 IN PDN 

The chemokine-receptor network has been 
widely implicated in both inflammatory pain condi-
tions and neuropathic pain. Chemokines are a sub-
set of cytokines responsible for attracting white 
blood cells to sites of inflammation through induced 
chemotaxis. C-C motif chemokine ligand 2 (CCL2) 
binds to C-C motif chemokine receptor type 2 
(CCR2), with a high level of affinity. Their interaction 
at the monocyte membrane is represented locally by 
a CCR2/CCL2 axis, creating a gradient with increas-
ing monocyte concentration in the direction of in-
flammatory sites. As a result of its influence on the 
migration of white blood cells, CCR2/CCL2 signal-
ing guides a number of key protective and destruc-
tive responses of the immune system (Hughes et al., 
2018).  

CCR2 is known to have two alternatively 
spliced transcript variants, CCR2A and CCR2B, 
which differ in the lengths of their cytoplasmic tails at 
the C-terminus. CCR2A consists of three exons, 
while CCR2B has an additional exon which is unique 
to that isoform. Alternative splicing describes the 
process wherein a single gene is assembled through 
different regulatory mechanisms to encode for mul-
tiple proteins. The precursor to mRNA, pre-mRNA, 
may contain exons that are omitted, shortened, or 
have higher sequence conservation as compared to 
other examples of mature mRNA. Conventional 

chemokine receptors, a family of chemokine-bind-
ing surface molecules, are often subject to alterna-
tive splicing. Like variants of CCR2, chemokine re-
ceptors CCR9, CXCR3, and CXCR4 have been spe-
cifically associated with changes in ligand-binding or 
signaling properties, which supports the idea that 
isoforms of CCR2 maintain altered properties.[5,18] G-
protein coupled receptors, such as CCR2, are de-
pendent on the amino acid sequence of their C-ter-
minal domain for intracellular signaling. Therefore, it 
is likely that functional differences between CCR2A 
and CCR2B will lead to their distinct signaling roles, 
despite the presence of a shared extracellular pro-
tein motif.[18]  

Preclinical studies have demonstrated the 
involvement of CCR2 and CCL2 in the pathophysiol-
ogy of neuropathic pain, with these models suggest-
ing that CCR2-antagonists can reduce painful symp-
toms.[1] A 2013 AstraZeneca study sought to evalu-
ate the efficacy and safety of the drug AZD2423 as a 
novel CCR2-antagonist in subjects with PDN.[9] CCL2 
plasma levels increased in a dosage-dependent 
manner, and mean monocyte levels declined by ap-
proximately 27%, indicating that AZD2423 inter-
acted with its intended target, CCR2. However, 
AstraZeneca’s study failed to show that AZD2423 al-
leviated primary and most secondary pain variables 
with greater efficacy than the placebo. We suggest 
that AZD2423 was found to be an ineffectual clinical 
treatment because it was not designed to cater to 
the distinct structural components of CCR2. To im-
prove the efficacy of medications like these, our re-
search has focused on understanding the levels of 
CCR2 variants in populations that suffer from PDN. 
Stratifying drug treatment based on the expression 
ratio of individuals’ CCR2 isoforms may enhance the 
efficacy of therapeutics in mitigating pain. From this 
data, we aim to decode the role of alternatively 
spliced CCR2 variants in the incidence of PDN and 
pathophysiology of neuropathic pain. We initially 
performed amplicon sequencing of PCR products to 
analyze the genetic variation in a specific region and 
confirm their genomic contents. Unexpectedly, we 
found evidence of additional alternative splicing 
sites, which implies the existence of a third isoform, 



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

CCR2C. The present study identifies CCR2 and char-
acterizes its presumed functional relationship with 
CCL2 following RT-PCR, gel electrophoresis, and 
analysis of amplicon sequencing results.  

 

2 METHODOLOGY  

Blood samples were obtained from 17 do-
nors; 11 were from individuals with diabetes and/or 
PDN, and 6 from individuals who were healthy. Each 
subject provided three 10 mL collection tubes of 
blood. We assigned each subject a letter from A 
through Q and marked the blood samples with their 
corresponding letter. Because monocytes are a di-
rect contributor to the neuroinflammatory model of 
DN, we were primarily interested in the buffy coat 
and plasma, as these components have the highest 
concentrations of white blood cells. We treated the 
collection tubes of buffy coat with RNAlater, a stabi-
lizing reagent that inactivates RNases and maintains 
the integrity of the RNA.  

We extracted RNA from the whole blood 
samples on the date of their arrival in an effort to re- 

duce degradation. First, we distributed the 5 mL of 
blood that was initially set aside into 200 μL test 
tubes, using the standard protocol from the NEB 
Monarch Total RNA mini-kit. The whole blood sam-
ples were lysed upon mixing with a 2X Monarch 
DNA/RNA Protection Reagent Concentrate. The 
whole blood RNA was then distributed into aliquots 
and stored at -80° C until further processing.   

 
HEPARINASE TREATMENT  

Because of the scarcity of the buffy coat layer and its 
viscosity, we experienced challenges extracting 
quantifiable levels of RNA, thus requiring that cDNA 
production in Reverse Transcription (RT) be maxi-
mally optimized. To improve cDNA yield, Hepa-
rinase I, a polysaccharide lyase, was added to the 
RNA before RT to cleave Heparin, an anticoagulant 
often used in blood sample processing. Heparin 
tends to be used to prevent the clotting and clump-
ing of cells, but it also inhibits the activity of the re-
verse transcriptase enzyme. Test tubes were pre-
pared containing RNA, Heparinase I, buffer, MgCl2, 

 

FIGURE 1: A comparison of CCR2’s previously recorded isoforms, which depicts that CCR2A lacks an additional exon.  Exons 1 
and 2 represent a conserved region between the variants, 67 base pairs (bp) and 992 bp long, respectively.  The last exon is 
2 bp longer in CCR2B than in CCR2A.  Primers listed within parentheses are situated at junctional sites, thus spanning two 
exons. 



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

and RNAse inhibitor (New England BioLabs rea-
gents). Some of these reagents were added in half-
volumes or less, with the remainder pipetted into the 
test tube after it was placed in a PTC-100 machine at 
25°C for a two hour-long incubation period.  

 
RT-PCR AND GEL ELECTROPHORESIS  

In designing isoform-specific primer pairs, we first 
developed primers which could distinguish CCR2A 
from CCR2B by binding to the regions unique to 
each variant. A two-letter nomenclature was devel-
oped, with the first letter referencing the forward pri-
mer and the second corresponding to the reverse. 
Because they are complementary to sequences on 
CCR2B-Exon 3, reverse primers F, G, and H, in com-
bination with a forward primer, can only successfully 
bind to and amplify CCR2B during PCR. However, 
conserved primer pairs able to bind to both CCR2A 
and CCR2B might still selectively amplify only a sin-
gle variant, depending on the resulting size of the 
PCR product. Any product greater than 1,000 base 
pairs (bp) long is generally longer than the polymer-
ization rate of the Taq polymerase enzyme can ac-
commodate under standard PCR extension times. In 
each cycle of PCR, 1 minute generally allows for up 
to 1 kb of the amplicon to be amplified, thus elimi-
nating larger products. For example, primer pair IN 
was previously confirmed by the author as a reliable 
CCR2A primer combination, yielding a PCR product 
267 bp long. IN can also bind to CCR2B, but the 
presence of the third exon creates an unlikely PCR 
product that stretches 1,464 bp; there is no current 
experimental data to support its specificity to 
CCR2B. CH and IH have both worked equally well as 
CCR2B-specific primer sets, likely because they bind 
to locations sitting relatively near each other. CH and 
IH isolate 309 and 222 bp PCR products in CCR2B, 
respectively. Both primer combinations PQ and VR 
have been validated as CCR2 internal controls be-
cause they bind to locations within the conserved re-
gion shared by each of the transcript variants, Exons 
1 and 2, producing identical amplicons in each. 

Following heparinase treatment, buffer, 
MgCl2, dNTPs, Random Hexamers, Oligo dT, and 
Reverse Transcriptase were incorporated into the 
RNA solute (SUPPLEMENTAL TABLE 1). The precise volumes 

of the reagents for PCR (SUPPLEMENTAL TABLE 2) tended 
to be more flexible because 1 to 3 primer sets can 
be tested against a patient sample, which slightly al-
ters the volume of water added to bring the master 
mix to 20 μL. The annealing step of PCR was consist-
ently run at 54°C for 30 seconds. To make the agar 
component of a gel, we added 0.35 g of Agarose 
and 25 mL of 1X TBE to a 50 mL flask and heated 
repeatedly until the solution was completely clear, 
with no pellets of agarose visible. The solution was 
poured into the tray of the gel box, solidified, and 
run at approximately 110 Volts until the bands trav-
elled two-thirds of the way to the opposite end of the 
gel. After being removed from the basin, the gel was 
stained with Ethidium Bromide for 20–30 minutes 
and photographed. 

All primers were designed using AmplifX 
and Oligo 7 software and ordered through Inte-
grated DNA Technologies. We carried out RT-PCR 
and gel electrophoresis for 12 primers pairs which 
had previously produced bands for CCR2A and 
CCR2B using reference RNA: CD, CJ, CL, CM, CN, 
CO, ID, IJ, IL, IM, IN, and IO. Amplicon sequencing 
was performed by GeneWiz, using next generation 
sequencing technology. 
 

3 RESULTS 
During our initial testing, we performed RT-

PCR and gel electrophoresis in order to establish re-
liable sets of primers for further quantification of 
CCR2 variants in patient samples. RT generated 
complementary DNA (cDNA), a copy of mRNA used 
as a template for PCR. In the following step, CCR2 
variant-specific primer pairs amplified CCR2A and 
CCR2B, creating millions of copies. These PCR prod-
ucts were evaluated using gel electrophoresis to 
confirm that their lengths matched the determined 
amplicon size of each primer combination. While gel 
electrophoresis is a reliable visual tool, amplicon se-
quencing was needed to positively identify each 
DNA sequence as a product of either CCR2A or 
CCR2B. Each lane of FIGURE 2 represents the contents 
of a CCR2A-specific primer combination. Clear 
bands can be seen at the expected amplicon lengths 
for all primer combinations except CF, whose at-
tributed PCR products were eliminated from the in- 



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

ventory stored for amplicon sequencing. Primer di-
mers appeared in all forward primer C-containing 
gel lanes below the 100 bp mark, often seen in Eth-
idium bromide-stained gels in the 30-50 bp range, 
resulting from primers attaching to each other.  

We also ran RT-PCR for other combinations 
that could be attributed to CCR2B (gel not pictured). 
There were a few instances of multiple bands in the 
case of CD, IM, IN, and CJ, which proved unusual 
due to the 1-minute cycle extension time during 
PCR, but not impossible for CCR2B-specific PCR 
products of approximately 1,000 bp. In theory, all 
four sets could bind to both variants, CCR2A and 
CCR2B. For the samples that presented the ex-
pected base pair lengths, our next step was to con- 

firm these results with amplicon sequencing. Follow-
ing high-throughput marker gene analysis, the am-
plicon sequences were read and totaled, represent-
ing a match to a predicted primer pair–generated 
PCR sequence.  

A major discrepancy was seen in the pro-
duced sequence alignment for primer set CD, which 
illustrated a missing piece of variant CCR2A nearly 
300 base pairs long. Amplicon sequencing of CD 
yielded four significant sequences which we referred 
to by their lengths in base pairs. 190, 356, and 208 
were a match to other chromosomes, while 115 cor-
responded to an ~180 gel band after accounting for 
the size of the forward and reverse primers.  
However, 115’s alignment to CCR2A was clearly dis- 

 

FIGURE 2: Gel depicting CD, CJ, CF, CM, CN, CO, ID, IJ, 
IL, IM, IN, and IO primer bands with plasma-extracted 
RNA as the template.  Each band corresponds to its “ex-
pected length in CCR2A” (TABLE 1), though no band ap-
pears for CF.  Primer pair CF may have been unsuccessful 
at binding to the cDNA if its melting temperature was 
outside of the range of the other sets, or within a region 
of secondary structure.  

 

TABLE 1: Expected primer pair lengths relative to isoform 
CCR2A, along with their approximate experimental band 
lengths determined from the published isoform’s DNA 
sequence.  

 



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

rupted, though there was no immediate evidence of 
alternate splicing against CCR2B (FIGURE 3). This find-
ing is highly irregular, considering that the primer 
pair must have successfully bound to the cDNA for a 
PCR product of any length to have been amplified. 

Primers CN, IN, CD, CJ CM, CO, ID IJ, IL, and 
IM produced segments that matched their expected 
CCR2A lengths. However, a few amplified fragments 
attributed to CN, IN, and ID were only partial 
matches to the entire expected sequence. For each 
primer combination, we analyzed the alignment of 
the sequences as compared the expected amplifica-
tion on either CCR2A, CCR2B, or both and gener-
ated dot matrix plots (not pictured) to better visual-
ize the accuracy of the predicted to the actual ampli- 

fication. At this stage, we intended to identify the ma-
jor sequences amplified, determine the primer se-
quences which might need to be trimmed or ex-
cluded from future analysis, and differentiate be-
tween those that were exclusive to either CCR2A or 
CCR2B.  

Of IN’s two significant sequences, IN 78 cor-
responded to various human and primate se-
quences other than CCR2, but IN 364 was an imper-
fect match to CCR2B, displaying possible evidence 
of alternative splicing due to the presence of multi-
ple GT-AG splicing sites (FIGURE 4). IN 364 exhibited 
other unexpected alignments, including a portion of 
CCR2B’s third unique exon situated within a region 
of CCR2A, as shown in FIGURE 5.  

 

FIGURE 3: The spaces highlighted in blue indicate expected base pairs absent from amplified CD 115, in relation to CCR2A.  
This missing sequence does not display the 5’-GT AG-3’ sequence characteristic of intron removal, as a result of alternative 
splicing. 



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

  

FIGURE 4: This alignment of IN 364 to CCR2B appears to be the result of alternate splicing, as evidenced by the yellow GT-
AG sequences.   

 

FIGURE 5: The base pairs highlighted in blue indicate a portion of the amplified sequence IN 364 that was not part of the 
predicted CCR2A sequence.    



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

FIGURE 6 identifies both an absent sequence 
from CCR2A and the addition of part of CCR2B, pro-
ducing a drastically changed sequence, shortened 
from the presence of additional splicing sites and yet 
incorporating parts of CCR2B-Exon 3.  
 

4 DISCUSSION  
Collectively, the sequence alignments com-

paring CD 115 and IN 364’s amplified products to 
our predicted sequences provide strong evidence of 
a third variant of the CCR2 gene. From our prelimi-
nary results, we observed unexpected alternative 
splicing and absent portions of the targeted isoform, 
revealing the existence of CCR2C. Due to the dis-
tinct inclusion of Exon 3 in CCR2B and parts of the 
same exon in CCR2C, the amino acid sequences of 
their C-terminus signaling region will be changed, 
likely causing functional differences in intracellular 

signaling to arise. CCR2C may have significance in 
the quantification of isoform expression levels be-
tween normal and diabetic patients. We propose 
that individuals’ expression profiles will translate to 
CCR2-antagonist responsiveness for AZD2423 and 
contribute to a genetic stratification approach for 
other PDN treatments.  

CCR2’s coding frame presents a long pep-
tide with the signature motif of an integral mem-
brane protein and a transmembrane domain. Alter-
native splicing shifts the translational frame, causing 
each of the three variants to represent a different 
reading frame. Despite lacking a fourth exon, 
CCR2A’s open reading frame is longer than 
CCR2B’s; thus, the variant maintains a longer car-
boxy terminal tail. CCR2C’s cytoplasmic tail is greatly 
truncated, suggesting that the open reading frame 
ends a few amino acids into the C terminal (FIGURE 7). 

FIGURE 6: In this region of the IN 364 sequence, the gene behaved primarily like CCR2A, though the blue highlights base 
pairs which should be present, and the yellow highlights a portion of CCR2B unexpectedly included in the alignment.  At 
the left is the proposed third isoform CCR2C.  This alignment suggests that the only difference between CCR2C and CCR2B 
is the contiguous nature of Exon 3 in CCR2B, and the removal of two introns from Exon 3 in CCR2C.  

 



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

 
The shortness of its carboxy tail could result 

in an effectively nonfunctional CCR2C isoform, the 
equivalent of a nonsense or stop mutation. CCR2C 
has a transmembrane and extracellular domain 
which appear identical to CCR2A and CCR2B, so 
CCR2C may become a sink, soaking up peptides or 
ligands that activate normal receptors. CCR2C 
would be similarly able to bind to CCL2, but this in-
teraction would not trigger an active, intracellular re-
sponse, reducing signal transduction. 

Assuming that CCR2C has the same 5’ se-
quence as CCR2A and CCR2B, either PQ or VR 
should, in theory, be able to target CCR2 equally 
well. As a result, the amplification of sequences us-
ing primers PQ and RV represents the total level of 
CCR2 expression, though there may be other undis-
covered isoforms of the CCR2 gene, which do not 
possess complete Exon 1 similarity. The next step 
with regards to primer design would be to synthe-
size primer sets that can reliably differentiate be-
tween CCR2B and CCR2C so that each isoform can 
be quantified separately. The PCR product of 
CCR2C should be shorter than CCR2B because of 
the removal of two introns, allowing us to discern be-
tween the variants. Two bands may be amplified, but 
given a distinctive difference in their lengths, gel 
electrophoresis bands should be clearly attributed 
back to CCR2B or CCR2C. We intend to determine 
CCR2 expression levels in order to understand the 
role of its isoforms in perpetuating nerve damage, 
pain, and/or loss of peripheral sensation.  

Although we began this study to identify ra-
tio differences in CCR2A/B in patient populations, 
amplicon sequencing results revealed distinct differ-
ences in sequences recovered from PCR, pointing to 
a third variant of CCR2. Alternative splicing of pre-
mRNA thus establishes three isoforms: A, B, and C. 
As a target of interest in prominent inflammatory and 
neuropathic pain conditions, antagonists of CCR2, 
such as AZD2423 have been clinically tested with 
some level of success.[9] However, CCR2 variant ex-
pression may offer insight into drug responsiveness, 
serving as a biological marker of PDN treatment effi-
cacy∎ 

 
5 ACKNOWLEDGEMENTS 
I would like to thank Dr. Lei Yu for his tireless support 
and guidance. It is because of his far-reaching intel-
lectual curiosity that I have had the privilege of work-
ing on this incredibly fulfilling project and have 
learned so much. I would also like to extend my 
thanks to fellow Neuroinflammation lab members 
Anika Patel and Madhuri Achanta, who not only con-
tributed data, but also carried the enthusiasm to fol-
low scientific leads like true detectives. 

 

 

FIGURE 7: Contrasts the extracellular protein sequences of 
CCR2’s variants.  The size of the protein’s cytoplasmic tail 
is a function of the reading frame’s positioning, which de-
termines the amino acids encoded by the gene.   



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

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Justine Soltys is a senior in the School of Arts and Sciences, majoring in Molecular Biology and 
Biochemistry, with a minor in history. Her research sits within the intersection between inflammation 
and neuropathic pain, under the guidance of Dr. Lei Yu at the Center for Alcohol Studies. At the Yu 
lab, she studies the isoform expression of CCR2’s alternatively spliced variants as a potential bi-
omarker and treatment target in Painful Diabetic Neuropathy. In the summer of 2021, she interned 
with the Discovery Toxicology- Non-Clinical Research and Development division of Bristol Myers-
Squibb to investigate calcium dysregulation of ryanodine receptor 2 (RYR2) in catecholaminergic 
polymorphic ventricular tachycardia (CPVT). The common thread underlying both projects is phar-
macogenetics, unique drug response influenced by genetic variations. After her undergraduate 
years, she hopes to obtain a PhD and contribute to pharmacology discovery and development. For 
questions, please contact her at: 
 JUSTINE.SOLTYS@RUTGERS.EDU

http://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-rna-purification-from-whole-blood
http://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-rna-purification-from-whole-blood
https://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf
https://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf
http://www.thermofisher.com/blog/biobanking/dna-purification-using-buffy-coat/
http://www.thermofisher.com/blog/biobanking/dna-purification-using-buffy-coat/


  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

7 SUPPLEMENTAL TABLES 
 
 

 
  

 
 
 
 
 
 
 

    
  

SUPPLEMENTAL TABLE 1: RT reagent table, establishing the 
standard reagent volumes per reaction.   

 

SUPPLEMENTAL TABLE 2: PCR reagent table, illustrating the 
variability of reagent ratios.   

*Can change relative to the volume of cDNA and number 
of primer sets per reaction tube. 

 



  ARESTY  RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III 
 
 
 

 
 

 

 

 

 

SUPPLEMENTAL TABLE 3: CCR2-specific primers, 
including their assigned letter, exon location, 
and forward or reverse nature. 

 

SUPPLEMENTAL TABLE  4: Amplicon sequence variant (ASV) table of major sequences, with the 
count noting how many times the sequence appeared within the PCR product sample for each 
primer pair.  The number of variants for each primer pair may indicate significant variability, or 
single polynucleotide polymorphisms (SNPs).  For example, the second and third CN variants 
are listed separately despite being the same length and having near-identical sequences be-
cause of a point mutation at the 27th base pair, where C has been changed to a T.  Similarly, 
the second variant of IN is a couple base pairs different from the first listed sequence.   

 


