1. A. Background
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that is
characterized by difficulty in communicating and forming relationships as well as
stereotyped restricted and repetitive patterns of behavior. ASD is typically present in
early childhood, with a rapidly expanding diagnosis that affects nearly 1 in 68 children.
Despite the prevalence of ASD, little is known about its cause. Evidence shows that
ASD is highly heritable; however, no single gene has been shown to affect more than 1%
of ASD cases.
Advances in both technology and research are beginning to explore the
importance and effects of non-protein coding RNA, which show that they may play an
integral role in regulating neurodevelopmental processes. Therefore, alterations in
noncoding RNA will cause defects in important developmental processes. Previous
research suggests that noncoding RNAs show altered expression in ASD brains.
Therefore it is likely that noncoding RNAs play a critical role in the disorder. The
function of most noncoding RNA is still unknown; therefore, determining the role they
play in altered ASD will help determine their greater functional role.
It is hypothesized that altered expression of regulatory noncoding RNA impacts
the molecular pathways leading to ASD. Through RNA sequencing on postmortem
human brains, we have identified a long noncoding RNA that is altered in ASD,
LINC01268, and we want to test it further to see the role it plays in the biological basis of
ASD. In order to test the functional properties of LINC01268, we wanted to overexpress
it in human neural progenitor cells. In order to do this, I worked on creating an

2. overexpression vector that will allow us to examine the biological consequences of
increased LINC01268 expression.
B. Research Methods
Five research methods were used throughout this project: cDNA synthesis,
polymerase chain reaction (PCR), gel electrophoresis, restriction enzyme digestion, and
ligation. First, the RNA was converted to cDNA through cDNA synthesis. Then PCR
was utilized to amplify LINC01268, generating millions of copies of LINC01268. Gel
electrophoresis was then used to test the size of the cDNA. LINC01268 is 2.7 kb long, so
we were looking to see a band that size. Once the appropriate size band was achieved,
the amplicon was sequenced to validate that the PCR product was LINC01268. After
validation, a restriction enzyme digestion was performed that allowed the ends to be
compatible for ligation, by cutting the LINC01268 sequence and making the ends
“sticky”. Once the amplicon was prepped a ligation was performed to fuse the
LINC01268 insert into the plasmid. The ligated products were then transformed into E.
Coli in order to amplify the overexpression vector.
C. Results and Discussion
During the project there were some delays. It was difficult making the right
primers that were specific to LINC01268 that would give the full-length transcript
because the 3’ end of the transcript has a very low GC content. To make the correct
primer an online program was utilized called “primer3”. To eliminate the problem with
the low GC content, 50 base pairs were taken off the 3’ end. Once the PCR worked, we
were able to isolate LINC01268. However, we then ran into troubles getting LINC01268
to ligate correctly to the pIRES2-AcGFP vector. After adjusting the ratios of plasmid and

3. insert as well as the ligation time and temperature, LINC01268 was able to ligate. We
performed transformations of the product into E. coli, which resulted in a product of the
correct size.
D. Conclusions and Future Directions
The importance of this study is to shed light on the molecular aspect of ASD,
about which little is known. Our results show that the noncoding RNA LINC01268 is
expressed at significantly higher levels in the postmortem brains of individuals with
ASD. We are now working to test the functional properties of this RNA transcript to
gain a better understanding of its molecular impact on ASD-relevant neuronal
architecture. Once this is achieved, it may be possible to develop better patient care,
including therapies and preventative care.
This study allows for follow up studies. Using the LINC01268 overexpression
plasmid that I created, the lab can conduct further tests. We can test the function of the
long noncoding RNA LINC01268 by overexpressing it with the plasmid in human neural
progenitor cells as they differentiate. We can determine the impact of LIN01268
overexpression on both global gene expression and neuronal morphology. In
complementary experiments, we can use small interfering RNA (siRNA) to knock down
the expression of LINC01268 and determine the impact of decreased LINC01268 on
human neuronal differentiation. These experiments will determine molecular mechanisms
that are relevant to ASD, and may lead to biologically based treatment options.