LAB REPORT

Student ID: 23047239

Word count: 1,850 excluding abstract and references

QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR): EXPRESSION OF

GENES IN TRANSGENIC ANIMALS RELEVANT TO DISORDERS

ABSTRACT

Measuring the expression levels of the brain-derived neurotrophic factor (BDNF) and Cyfip1
genes in wildtype and Cyfip1 heterozygous deletion rat models is the main goal of the practical.
The goal of this setup is to replicate the genetic circumstances linked to mental illnesses including
autism spectrum disorder (ASD) and schizophrenia.
A number of crucial procedures are involved in the practical: RNA is extracted and isolated from
brain tissues, RNA is then converted to complementary DNA (cDNA), and qPCR is set up using
certain primers for the target genes. The procedure for making the Master Mix, the particulars of
the heat cycling procedure, and the following qPCR data analysis to ascertain the relative gene
expression levels are all described in the publication. Because of its sensitivity and
quantifiability—both of which are critical for comparing gene expression levels among genetic
models—qPCR is used here. The significance of QuantiTect SYBR Green, a double-stranded DNA
binding dye, in the identification and measurement of DNA during the qPCR procedure, is
emphasized in the text. The practical also incorporates instructional elements, such as activities
that explain the scientific rationale for the use of transgenic mice in research, the particular uses
and benefits of qPCR, and the applicability of the Cyfip1 deletion model in simulating mental
disorders in humans. These tasks are intended to improve one's comprehension of the experiments'
technical details as well as their larger scientific context.

INTRODUCTION:
The primary goal of the project is to look at how the Cyfip1 gene may be involved in mental
illnesses including intellectual disability (ID), schizophrenia, and autism spectrum disorder (ASD).
These diseases are known to be linked to the Cyfip1 gene, especially when it is deleted or
duplicated on human chromosome 15 (Ellenbroek and Youn, 2016). Researchers have developed
genetic rodent models, such as Cyfip1-deleted rat and mouse models produced by CRISPR-Cas9,
to better understand the genetic pathways underlying these illnesses. These models replicate the
genetic alterations seen in human disorders (Bustin et al., 2013).
In this work, gene expression in these rodent models is measured using quantitative polymerase
chain reaction, or qPCR. qPCR is a potent molecular test that combines conventional PCR methods
with QuantiTect SYBR Green, a double-stranded DNA binding dye (Dheda et al., 2004). During
the PCR extension phase, this dye intercalates into double-stranded DNA sequences, producing a
strong fluorescent signal that the thermocycler detects (Martinowich, Manji and Lu, 2007). This
makes it possible for scientists to measure the quantity of double-stranded DNA that is there, giving
important details on the amounts of gene expression. In order to better understand the molecular
mechanisms behind psychiatric diseases, researchers can examine the impact of Cyfip1 deletions
or duplications on gene expression in the mouse models by the use of qPCR in this investigation.
MATERIALS AND METHODS
Part I of the experiment: RNA to cDNA conversions (about 60 minutes)
1. RNA extraction from Cyfip1 and WT After completing the het rat brain tissue, RNA was
isolated using the RNeasy Qiagen Kit. The process of transforming RNA into single-
stranded complementary DNA (cDNA) comes next.
2. Put the UltraScript RTase and 5x cDNA Synthesis Mix (qPCRBIO cDNA Synthesis Kit,
PCR Biosystems, PB30.11-10) on ice.
3. After thawing the ingredients, give the cDNA Synthesis Mix a quick vortex. Do not vortex
the enzyme UltraScript RTase.
4. For the two reactions, make the Master Mix in a single tube according to the given order:
a. 8 µL of 5x cDNA Synthesis Mix
b. 20 times RTase: 2 µL
5. After properly mixing the Master Mix, divide the mixture into two 0.2 mL PCR tubes by
aliquoting 5 µL of each. In order to generate two distinct cDNA samples, mark these tubes
with the initials WT and Het.
6. Fill the WT RNA template tube with water in accordance with the guidelines given:
a. Add 13.0 µL of PCR-grade water to WT_A.
b. Add 13.1 µL of PCR-grade water for WT_B or WT_C.
7. For the Heterozygous RNA template tube, repeat these steps again, adding water as
directed:
a. Add 12.4 µL of PCR-grade water to Het_A.
b. Add 13.0 µL of PCR-grade water to Het_B.
c. Add 12.7 µL of PCR-grade water to Het_C.
8. This yields a total volume of 15 µL, which is predicted to give 200 ng (or 0.2 µg) of RNA
needed for the following qPCR step.
9. Gently stir the ingredients by aspiration.
10. Utilizing the "RT PCR Biosystems" thermal cycler, incubate and denaturize the two
samples for thirty minutes at 42°C, then for ten minutes at 85°C.
11. The freshly generated cDNA will be 200 ng total in 20 µL upon completion, yielding a 10
ng/µL concentration.
12. As instructed, set up responses for Part II or carry out other tasks during this incubation
time.
13. Take the tubes out of the heat cycler and put the freshly made cDNA on ice.

Part II of the experiment: qPCR setup (about 30 minutes)

 1. About fifteen minutes before to preparation time, thaw the six primer tubes and QuantiTect
SYBR Green (Qiagen, 204143) on ice.
2. Spin down the QuantiTect SYBR Green after briefly vortexing it.
3. Prepare three distinct Master Mixes (MM) in tubes with labels, one for each of the three
genes (Gapdh, Cyfip1, and BDNF), including forward and reverse primers.
4. Each gene's two response amounts are as follows:
a. 20 µL of QuantiTect SYBR Green
b. 1.6 µL of forward primer (10 µM)
c. 1.6 µL of reverse primer (10 µM)
d. 12.8 µL of water
5. Pipette 18 µL of each Master Mix into the wells of the six connected tube strips, starting
from the leftmost right: Gapdh MM, Gapdh MM, Cyfip1 MM, Cyfip1 MM, BDNF MM,
and BDNF MM. Vortex each Master Mix.
6. To make the strips' orientation obvious, label them.
7. Fill each of the six tubes with 2 µL of the pre-prepared cDNA in turns, so that each tube
has a final capacity of 20 µL.

qPCR amplification in Part III of the experiment (about 1.5 hours)

We use the HotStarTaq DNA Polymerase that comes included in the QuantiTect SYBR green kit
for this step. Due to a chemical modification, this polymerase is inactive until the first heat
stimulation step at the start of the PCR. These characteristic stops primer dimers and non-
specifically annealed primers from extending during the PCR setup and first cycles, which can
happen at low temperatures. In today's thermal-cycler program, HotStarTaq DNA Polymerase is
activated by a 15-minute incubation at 95°C.

The use of HotStarTaq eliminates the need to keep responses on ice.

The following were our PCR cycling settings:
• One cycle lasting ten minutes at 95°C
• 40 cycles, including 15 seconds of denaturation at 95°C
• One minute of annealing and elongation at 60°C
 RESULTS
1. Amplification Curve

Fig. 1 Amplification curve following qPCR run

The graph's colors correspond to each sample, which are:
1st sample (Gapdh WT) – Purple
2nd sample (Gapdh Het) – Not detected
3rd sample (Cyfip WT) – Not detected
4th sample (Cyfip Het) – Not detected
5th sample (BDNF WT) – Yellow
6th sample (BDNF Het): Not detected
 Table 1. Result obtained as tabular form after the qPCR run
Table 1 displays the melting temperatures and cycle threshold values for each sample, starting with
the first specimen from wells D1 through D6. Wells D2, D3, D4, and D6 did not provide a cycle
threshold at 84°C among the data that were collected.

2. Calculations below are being followed and done according to the example presentation
slides
Cyfip -
TE – HE = 26.11 - 20.99 = 5.12
So, 5.12 is △CTE

TC - HC = 24.12 - 20.32 = 3.8

This is △CTC

△△CT = △CTE - △CTC

5.12 - 3.8 = 1.32
Hence, Expression in fold change = 2^-1.32 = 0.4005349
So, in percentage = 1 - 0.4005349 X 100 = 59.94%
Hence, there is a 59.94% decrease in gene expression in case of Cyfip
BDNF –
TE is 34.53 and TC is 25.81
TE - HE = 34.52-20.99 = 13.54 (△CTE)
TC - HC = 25.81 - 20.32 = 5.49 (△CTC)
△CT = 13.54 - 5.49 = 8.05
Expression in fold change = 2^-8.05 = 0.0037731887848
In %age = 99.62%
Hence, in the case of BDNF, there is a decrease in gene expression of 99.62%

3.

Fig 2. Graph on relative expression for Cyfip 1 and BDNF

4. Fig 3. Graphical presentation

Table 2. Primer Pairs

 DISCUSSION
1. The use of distinct primers in qPCR provides benefits such as simultaneous
quantification of numerous targets, identification of isoforms or splice variants, and
targeting of several gene areas for complete coverage. Primer’s dimers, on the other
hand, are non-specific products that might arise and must be minimized by careful
design and optimization (Suzuki, Higgins and Crawford, 2000). This entails employing
the proper annealing temperatures and making sure primer sequences do not self-
complementarity. Including a no-template control (NTC) also aids in the detection of
non-specific amplification and interference.

2. Because Cyfip1 is involved in neurodevelopmental processes and synaptic function, it

is useful in understanding a variety of neurological illnesses. A crucial part of the
WAVE regulatory complex, Cyfip1 controls the cytoskeletal structure and actin
dynamics that are essential for synaptic plasticity and neural connection.
Neurodevelopmental illnesses such as autism spectrum disorder (ASD), schizophrenia,
and intellectual difficulties have been linked to dysregulation of Cyfip1. Researchers
can look into the molecular mechanisms causing these illnesses and find possible
targets for treatment by examining animals with Cyfip1 deficiency. Additionally,
comprehending Cyfip1's involvement in synaptic function may shed light on more
general elements of neuron physiology and aid in the creation of cutting-edge
therapeutic approaches for neurological diseases (Peça et al., 2011).

3. Technological developments such as accurate primer sets, digital PCR methods, and
high-throughput qPCR systems have greatly improved gene expression analysis.
Numerous genes may be examined simultaneously on these platforms, and non-specific
amplification is decreased thanks to better primer design. Accuracy is improved by
digital PCR, which allows for absolute quantification without standard curves. The
understanding of gene control and cellular heterogeneity has been revolutionized by
single-cell RNA sequencing. When taken as a whole, these developments spur ongoing
development in gene expression analysis, expanding our knowledge of biological
systems and illnesses.

The implementation of the experimental technique successfully discerns variations in Cyfip1 gene
expression, as demonstrated by the decreased Cq values between the heterozygous (Het) and wild-
type (WT) samples (Keifer and Summers, 2016). This suggests that the decreased Cyfip1
expression in the Het sample was successfully detected. Furthermore, knowledge on BDNF
expression can advance understanding, particularly with regard to mental illnesses. Any decrease
in BDNF expression in the Het sample, if BDNF levels were assessed in conjunction with Cyfip1,
may indicate a link between Cyfip1 deletion and changed BDNF levels. This correlation suggests
that dysregulated neurotrophic pathways—where dysregulation of BDNF is observed—may be
involved in disorders such as depression and schizophrenia. These conclusions are purely
speculative, though, in the absence of accurate BDNF expression data.
All things considered, it seems that the experimental strategy was successful in identifying
variations in Cyfip1 gene expression between the WT and Het samples. If accessible, the BDNF
expression data may offer more information about the consequences of Cyfip1 deletion for
neurodevelopmental processes and the pathophysiology of psychiatric disorders.

The observed decrease in Cyfip1 gene expression in the heterozygous (Het) sample compared to
the wild-type (WT) sample may stem from several factors (Bustin et al., 2009). Firstly, genetic
deletion or mutation in one allele of Cyfip1 in the Het sample reduces its expression relative to the
two intact alleles in the WT sample. Dosage effects also play a role, with one functional allele in
the Het sample resulting in lower Cyfip1 mRNA abundance. Additionally, disruptions in regulatory
mechanisms due to the genetic alteration may further decrease expression. Discrepancies in RNA
quality or quantity between samples can affect cDNA synthesis and qPCR amplification. Finally,
inherent experimental variability, including pipetting errors and primer effectiveness, can influence
results despite careful methodology and controls (Wen et al., 2014).

Features of Alternative Primers: The alternative primers utilized in this experiment probably
have a number of important features, including:
Specificity: To guarantee specific amplification and precise quantification, primers are made to
target particular sections of the target gene (such as Cyfip1, BDNF, and Gapdh).
Efficiency: For accurate and repeatable qPCR findings, primers should have a high amplification
efficiency. The usual method for assessing this efficiency is standard curve analysis.
Consistency: Across several experimental replicates and circumstances, primers should yield
findings that are repeatable and consistent.
Absence of primer-dimers: Primers shouldn't combine to generate non-specific amplification
products or primer-dimers, as they might impede precise target gene quantification.
Compatibility: The buffer composition and annealing temperatures used in the experiment should
be acceptable with the primers.

For precise gene expression normalization in qPCR assays, a housekeeping gene such as Gapdh
is essential. Gapdh is extensively utilized because of its high mRNA abundance, steady expression
in a variety of tissues and cell types, and participation in essential metabolic processes including
glycolysis. It is a trustworthy reference for determining targeted gene expression levels in relation
to a steady baseline because of its stability and widely recognized validation.
 ACKNOWLEDGMENT
I extend my sincere gratitude to Keele University for offering significant input into our study. I am
very grateful to Dr. Simon Trent for his wonderful technical support. I would also like to thank
Keele University and Dr. Marcelo for their insightful conversations. Finally, I express my gratitude
to my friends and colleagues for their continuous encouragement and support during this endeavor.

REFERENCES
• Bustin, S.A., Benes, V., Garson, J., Hellemans, J., Huggett, J., Kubista, M., Mueller, R.,
Nolan, T., Pfaffl, M.W., Shipley, G., Wittwer, C.T., Schjerling, P., Day, P.J., Abreu, M.,
Aguado, B., Beaulieu, J.-F., Beckers, A., Bogaert, S., Browne, J.A. and Carrasco-Ramiro,
F. (2013). The need for transparency and good practices in the qPCR literature. Nature
Methods, 10(11), pp.1063–1067. doi: https://doi.org/10.1038/nmeth.2697

• Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R.,
Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. and Wittwer, C.T. (2009). The
MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR
Experiments. Clinical Chemistry, [online] 55(4), pp.611–622. doi:
https://doi.org/10.1373/clinchem.2008.112797

• Dheda, K., Huggett, J.F., Bustin, S.A., Johnson, M.A., Rook, G. and Zumla, A. (2004).
Validation of housekeeping genes for normalizing RNA expression in real-time PCR.
BioTechniques, 37(1), pp.112–119. doi: https://doi.org/10.2144/04371rr03

• Ellenbroek, B. and Youn, J. (2016). Rodent models in neuroscience research: is it a rat

race? Disease Models & Mechanisms, [online] 9(10), pp.1079–1087. doi:
https://doi.org/10.1242/dmm.026120

• Keifer, J. and Summers, C.H. (2016). Putting the ‘Biology’ Back into ‘Neurobiology’: The
Strength of Diversity in Animal Model Systems for Neuroscience Research. Frontiers in
Systems Neuroscience, 10. doi: https://doi.org/10.3389/fnsys.2016.00069

• Martinowich, K., Manji, H. and Lu, B. (2007). New insights into BDNF function in
depression and anxiety. Nature Neuroscience, [online] 10(9), pp.1089–1093. doi:
https://doi.org/10.1038/nn1971

• Peça, J., Feliciano, C., Ting, J.T., Wang, W., Wells, M.F., Venkatraman, T.N., Lascola, C.D.,
Fu, Z. and Feng, G. (2011). Shank3 mutant mice display autistic-like behaviours and
striatal dysfunction. Nature, [online] 472(7344), pp.437–442. doi:
https://doi.org/10.1038/nature09965
• Suzuki, T., Higgins, P.J. and Crawford, D.R. (2000). Control Selection for RNA
Quantitation. BioTechniques, 29(2), pp.332–337. doi: https://doi.org/10.2144/00292rv02

• Wen, Z., Nguyen, H.N., Guo, Z., Lalli, M.A., Wang, X., Su, Y., Kim, N.-S., Yoon, K.-J.,
Shin, J., Zhang, C., Makri, G., Nauen, D., Yu, H., Guzman, E., Chiang, C.-H., Yoritomo,
N., Kaibuchi, K., Zou, J., Christian, K.M. and Cheng, L. (2014). Synaptic dysregulation in
a human iPS cell model of mental disorders. Nature, 515(7527), pp.414–418. doi:
https://doi.org/10.1038/nature13716