BIO/350 v5

Gene Expression Unit: Use Sequencing to

Unveil a Gene Linked to Obesity Lab
Reporting Worksheet
In science, reporting what has been done in a laboratory setting is incredibly important for communicating,
replicating, and validating findings. However, writing scientific reports can be a little overwhelming. There
is a set of agreed-upon components that the scientific community requires when reporting scientific
research. Answer the following questions to describe what occurred during the lab you conducted in
Labster. Be sure to use complete sentences and descriptions that fully represent what you experienced.
Writing a lab report is less about being correct or incorrect than it is accurately reporting what happened
and why. So, do not worry about reporting data that might seem counterintuitive or unexpected. Focus on
clearly communicating what you did and what you observed.
Write your answers on a new line.

Title
1. What was the title of the lab you completed?
Gene expression: Use sequencing to unveil a gene linked to obesity.

Topic
2. What was the subject you were trying to understand better in the lab?
The subject that I was trying to better understand during this lab was learning how to prepare
samples for next generation sequencing, comprehend the principles behind the next generation
sequencing technique, and perform qPCR experiments.

Background Information
3. What information from the textbook and classroom is relevant for the subject you were trying to
gain a better understand of in the lab? Identify the concepts and explain how they are related to
the lab topic.
Everybody has DNA, but uniquely. The book covers the foundations of DNA replication, DNA
structure, DNA sequencing, DNA replication in prokaryotes and eukaryotes, DNA repair, genetic
code, and gene control. It covers comprehending next generation sequencing and doing data
analysis from an mRNA sample, analyzes the produced sequencing data, understands of how
mRNA connects to gene expression and its impact on obesity, perform qPCR, and cDNA
synthesis throughout the lab. Learning how to sequence DNA and assess the expression levels of
a certain gene and identifying the two live pig mRNA samples was the aim in order to find which
genes lead to obesity.
4. During the lab, what information from the theory section provided additional background
information about the subject? (To review the theory section, launch the lab and click the Theory
tab on the top of the data pad). Identify the concepts and explain how they are related to the lab
topic.
Short DNA molecules sequenced simultaneously and subsequently linked to their reference
sequence enable next generation sequencing (NGS), therefore completing the DNA sequence of
the genome. To be distinguished during sequencing, the four nucleotides are tagged with a
particular fluorophore. Adenine is orange; thymine is green, cytosine is blue, guanine is red.
Three NGS data analysis—primary, secondary, and tertiary—as well as other analysis exist.

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 Gene Expression Unit: Use Sequencing to Unveil a Gene Linked to Obesity Lab Reporting Worksheet
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Trimming the tag and adapters comes first; during the second phase, the analysis compiles all the
short DNAA sequences to decipher the sequence data. Last phase notes the gene expression
levels and evaluates the data.
5. Most scientific observation involves examining phenomena or processes. What phenomenon or
process were you observing in the lab? What were you able to change and explore? What did the
simulation not allow to change?
During DNA sequence and gene expression, it is crucial to apply appropriate PCR mixtures. In this
case, we sequence DNA and use suitable data analysis and NGS and qPCR to exclude any genes,
so determining whether the genes could be over expressed in obese pigs. We investigated every
method and approach that is original and pertinent in determining the gene connection causing
obesity. For instance, preparing the cDNA synthesis and observing where the primers bind to the
tail, reverse transcriptase, NGS utilizing the obtained sample, add adaptors to the ends of the
DNA, generates RNA clusters. Mixing the wrong DNA samples is what the lab forbade changing.

Method
Describing what you did during a lab supports other scientists in replicating your work. It is through this
consistent replication that scientists are able to see repeating patterns and develop ideas that help move
science forward. When you discuss your observations, in a later section, you will have to describe, in
detail, what you did. You may also have to describe what choices you made, why you made them, and
any concerns about things that occurred that were unexpected. To have enough information to do this,
you need to keep very detailed notes. What doesn’t seem important in the moment may end up being
something that explains your findings later. A benefit of conducting virtual labs when learning science, is
that many potential errors are controlled for you. The virtual lab environment often will alert you if
something is not going the way it should. This does not occur in non-virtual settings. The virtual lab setting
can be very helpful to learners for this reason. However, we still need to practice documenting so those
skills are practiced for the lab experiences when technology will not be there as a coach.
6. You have already described the phenomenon or process you studied in the lab in the previous
section. Now, take some time to fully describe the steps you took during the lab. Do not include
the process of you logging into the lab in your description. For this virtual lab, a short, high-level
summary will suffice.
Synthesizing cDNA by mixing it with mRNA in a PCR tube comes first; then, put the tube in the
PCR machine to initiate the synthesis of cDNA. The magnetic bead purification technique causes
the cDNA strand to break apart. Adenine is added to the purified DNA by means of an adaptor
sequence introduced at the ends of cDNA and thermoshaker mixing action. Adenylated DNA
separates from the buffer and the enzymes. Mixing the adaptable and adenylated DNA in a 1.5 mL
tube, the thermoshaker is intubated. PCR is being done and amplify cDNA strands following
incubation. Genes are then examined from the sequencing process once it is finished. After that,
present genes from the samples are sought for using RNA sequencing. We check findings and
guarantee there is no contamination—that might tamper with the DNA template—by using a
negative control.
7. Describe some of the observations you made. What did you write down or keep track of? What
did each of your senses observe during the lab process? What did you see (e.g., changes in
colors, movement, shapes, sizes, patterns)? What, if anything, did you measure? What did you
hear (e.g., sounds from reactions, collisions, error messages)? What did your lab character
touch? Did you notice anything that seemed unexpected? Did you notice anything that you did
not expect to observe?
What I witnessed were the data and results we saw in the lab, such as the newly produced DNA
strand that used reverse transcriptase enzyme to use mRNA as a template. During the visual, the
adenosis was deposited on the 3' end of the DNA, and the enzyme attached to the DNA and added
another adenine base to the 3' end, which then overhangs at the end, allowing it to bind to the
adaptor. After performing the PCR and amplifying the cDNA strands, the strands will employ the
primers to bind to their unique complementary adapter sequence under high temperatures. After

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 Gene Expression Unit: Use Sequencing to Unveil a Gene Linked to Obesity Lab Reporting Worksheet
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the amplified PCR bridge, an elusion buffer is used to wash away a cluster. When performing
qPCR on a specific gene, a reference gene is required to ensure that it is expressed uniformly
across all cell types. The goal of this reference gene is to normalize the amount of beginning
material in a sample. The leptin gene was overexpressed in obese pigs, which links leptin,
obesity, and adipose tissue together.
8. Which parts of the lab required you to think more than others and required more time? Which
parts were simple and completed easily?
This lab was very interactive and hands on working with the different DNA samples. I felt that it
required time to make sure that the samples were accurately mixed correctly and placed in the
correct machine either the PCR machine or thermoshaker, I felt that the instructions were very
clear and the theory helped out quite a bit however at first I didn’t realize that if you hovered over
the vials, it told you what sample it was. After I found that out, the rest of the lab was pretty
straightforward.

Observations
Many lessons learned from scientific research come from the reporting and analysis of data and
observations. This part of scientific reporting requires detailed descriptions of technical information and
observations, as well as high-level synthesis of information. High-level synthesis requires a mastery of
foundational content in the related scientific field and a complementary mastery in some field of
quantitative and/or qualitative analysis. For this report, let’s focus on big picture patterns.
9. What did you notice about the phenomenon or process you explored?
I find that starting the whole process depends on building a DNA. We had to make sure our DNA
was sufficient for sequencing. Moreover important for DNA is temperature. We found that the
cDNA synthesis guarantees strong temperature separation of the molecules. High temperatures
denature the double stranded DNA thereby transforming one strand. This was clear to us from
using some samples. For instance, running the PCR tube with the cDNA synthesis mix through
the PCR machine with mRNA.
10. Describe any information about the phenomenon or process that you learned.
Everything in the lab seemed quite hands-on and very new for me to learn. NGS or qPCR is one of
the few methods available to evaluate gene expression levels. Testing models of pigs as their
body composition and genome reflect human nature the closest thing available. Magnetic beads
helped extract the polymerase and other PCR reagents, therefore finishing the preparation
process. Attached with a particular fluorophore are the nucleotides. One 3' cap only lets one
nucleotide be connected. Every nucleotide has a fluorophore that lets one view the sequence.
Green tags thymine; cytosine with blue; guanine with red; adenosine with orange.

Discussion
The discussion section is used to explain why things might have happened the way that they did in your
research. Here, scientists describe any potential anomalies or mistakes and why they think they may
have occurred.
11. During your lab, what happened that might have had an impact on the accuracy of your
observations? Did the simulation alert you that an error was occurring? If so, how did you resolve
it?
The simulation permitted no inaccuracies, particularly when combining several samples. If you
selected the incorrect sample mixes, the laboratory would have informed you to obtain another
tube and mix the appropriate samples. The possibility of unnotified mixing would impact the
results in identifying the gene associated with obese pigs.
12. The discussion section also is used to summarize big ideas from the lab. What were the
important learnings about the phenomenon or process from the lab?

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 Gene Expression Unit: Use Sequencing to Unveil a Gene Linked to Obesity Lab Reporting Worksheet
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Important lessons about the procedures and methods we applied throughout the lab are that,
in NGS to find the gene linked to obesity, every step is vital; in preparation of qPCR and
melting curves. The first stage is cDNA synthesis, cDNA and mRNA reversal with reverse
transcriptase, add adaptor sequence, run PCR to enhance the cDNA strand and make sure
there are enough strands to perform sequencing, trim off adaptors, analyze and check on
gene expression levels. Leptin reduces appetite; in obesity this is unregulated. Obesity affects
leptin. This gene expression in the mRNA samples by qPCR is verified. The gene expression
generates the RNA and proteins from the gene by transforming it into a cell. Protein and RNA
synthesis occurs.

Conclusion
The conclusion section of a lab report describes how the learnings from the lab research fit in to prior
scientific knowledge. This is done by comparing new information to previously known information that was
identified in the section of your report that discusses background information.
Review the background information section of your report from above and describe how the results of
your lab compare to the information you discussed before.

13. After scientists have identified how the new knowledge fits into the old knowledge, they discuss
the implications of the new information for moving forward. In this class, the purpose of study is to
learn some foundational science ideas represented by the course learning outcomes. Review the
course learning outcome aligned to this lab in the assignment directions in Blackboard. How is
the information from this lab related to the course learning outcome? What knowledge has the lab
supported you with learning that is related to this course learning outcome?
Seeing the effect genes have on inheritance and genetic expression is the course learning
outcome. Examining lean and obese pig samples along with an mRNA sample helps us to
understand the relationship between genes and obesity in the lab. We gained knowledge on DNA
sequencing and searching for certain genes expressed in fat pigs. Identifying particular features—
obese genes in this case—we analyzed and separate out these particular genes using NGS and
qPCR.
14. Following scientific research, scientists usually come up with new questions that result from what
they learned. These new questions often end up leading to new research in the future. What
additional scientific things do you wonder about after completing and writing about your lab
experience?

We discovered the relationship between leptin and obesity by means of sequencing DNA
and applying qPCR. Is this the same PCR test applied in the same method used in
coronavirus testing?

When it comes to environmental factors, we know that temperature may effect the gene
expression but what about light or exposure to toxins?

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