LAB 8

GEL
ELECTROPHORESIS,
M I C R O A R R A Y,
R N A S E Q A N A LY S I S

SCB252 Fundamentals of
Biotechniques
Objective

•To practice casting a gel and interpret the result

•To understand the microarray and RNA-seq and their difference
•To practice analysing mRNA expression dataset with Galaxy
•To be able to interpret the visulization
 Agarose Gel
Analysis
Agarose gel analysis enables quick and
easy quantification of DNA, especially for
small DNA fragments (such as PCR
products). As little as 20 ng of DNA can be
detected by agarose gel electrophoresis with
ethidium bromide staining. The DNA sample
is run on an agarose gel alongside known
amounts of DNA of a similar size.
The amount of sample DNA loaded can be
estimated by comparison of the band
intensity with the standards using a scanner
or imaging system. Standards of roughly the
same size as the fragment of interest should
be used to ensure a reliable estimation of
the DNA quantity since large fragments
intercalate more dye than small fragments
In a gel, agarose forms a mesh that functions as a molecular
sieve. Gels with higher percentages of agarose create a tighter
mesh with smaller pores. In figure 1, a 1% gel (left) exhibits
significantly larger pores compared to a 2.9% gel (right).
Gel percentage
Generally, higher percentage agarose gels are more effective for separating smaller
DNA fragments, whereas lower percentage gels are preferable for separating larger
DNA fragments. The table below summarizes common agarose gel percentages and
the corresponding range of DNA fragment sizes that can be effectively separated:

Agarose Gel %: Range of Effective Separation (bp)

Other factors that influence band resolution
1. Voltage and Run Time: The voltage applied during electrophoresis and the duration of the run impact
band resolution. Higher voltages can lead to faster migration of DNA fragments but may result in
increased heating and band smearing. Longer run times can improve resolution but may also cause
diffusion and broadening of bands.
2. Buffer Composition: The type and concentration of the electrophoresis buffer used can affect band
resolution. Buffers such as TAE (Tris-acetate-EDTA) or TBE (Tris-borate-EDTA) provide stable pH and
ionic strength, which offers better resolution for smaller DNA fragments.
3. Gel Thickness: The thickness of the gel can influence resolution. Thinner gels allow for better heat
dissipation and more efficient separation of DNA fragments compared to thicker gels, which may
experience uneven heating and slower migration rates.
4. DNA Loading Amount: Overloading the gel with DNA samples can lead to overcrowded bands and
poor resolution. Optimizing the amount and volume of DNA loaded onto the gel ensures clear and
distinct bands.
5. Staining and Visualization: The choice of DNA stain and the method of gel visualization (UV
transillumination, fluorescence imaging, etc.) can affect the clarity and visibility of DNA bands. Proper
staining and imaging techniques are essential for accurate band analysis.
6. Quality of DNA Samples: The quality and purity of DNA samples used in electrophoresis influence
band resolution. Contaminants or degraded DNA can result in smearing or indistinct bands.
 1 bucket of ice( thaw below 3 items on ice)
8 PCR samples from previous lab 7
2 pBE322/BstNl Marker
2 5X loading dye

1 20xTBE buffer
2 250ml flask
2 SeeGreen agarose tab
L AB EXERCISE I 2 2-20ul micropipette with 1 box of P20-200
GEL 2 100ml graduate cylinder

ELECTROPHORESIS 2 bottle of 500ml Distiled water

2 gel apparatus with 9 gel comb with 9wells
2 BlueGel apparatus
 Methods
Making the 2% gel:
1.Add 1 SeeGreen all-in-one agarose tab, 20ml Distilled water to 250ml flask, swirl to mix and
microwave for 30s.
2.Place the flask on bench to cool down for 1min, then pour the gel into the gel casting tray.
3.Insert the 9 well comb into the gel casting tray, each well holds up to 20ul samples.
4.Let gel stand for ~10 minutes until completely set. For faster set time place the casting platform
with the gel in a refrigerator. Do not disturb the gel during this time.
5.While waiting for the gel, prepare the DNA samples for loading.
6.To each PCR sample tube, add 12.5ul 5X loading dye and mix by pipetting in and out.
7.After the gel has solidified, remove comb/s gently by pulling straight upward.
8.Remove the gel tray from the casting platform. If a small amount of gel has formed underneath the
gel tray, wipe it off and discard it.
9.Place the gel tray containing a gel in the buffer chamber and place the buffer chamber inside the
blueGelTM base. The wells should be closest to the (-) end.
10.Add 40 ml of 1X TBE buffer in the buffer chamber. The buffer should just cover the agarose gel.
11.Remove air bubbles (if any) trapped between the gel and the gel tray, or between the gel tray
and the buffer chamber.
Load DNA samples to the wells and run the gel.

1.Load 15ul pBR322/BstNl marker to the 1st line of the

gel.
2.Load 15ul of each PCR samples to the rest of wells.
3.Run the gel for approximately 40mins.
4.Observe the result of the gel by turning on the UV
light.
5.Take picture of result and interpret the result on lab
notebook.
 Sample WT WT Vg Vg Eb Eb 2
2
Primer Eb Vg Eb Vg Eb Vg Eb
Vg
 Introduction to
Microarray
Microarray technology is a common lab technique
that involves attaching a large array of known
nucleic acid fragments, ranging from thousands
to millions, onto a solid surface known as a
“chip”. This chip is then exposed to DNA or RNA
extracted from a sample under study, such as
cells or tissues. The interaction between the
sample and the fragments immobilized on the
chip, through complementary base pairing,
generates light via fluorescence. This light can be
detected using a specific machine. Microarray
technology has a wide range of applications in
both research and clinical studies, including the
measurement of gene expression and the
identification of specific DNA sequences, such as
single-nucleotide polymorphisms (SNPs).
What’s inside the well:
What color will
the well show if
no DNA bonds to
the probe?
RNA-seq (RNA sequencing)
RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that
uses next-generation sequencing to reveal the presence and quantity of RNA
molecules in a biological sample, providing a snapshot of gene expression in
the sample, also known as transcriptome.

RNA-Seq is transforming our understanding of the transcriptome. As a highly

precise and sensitive method for quantifying expression across the
transcriptome, it offers researchers insights into previously unseen changes
that occur in various states of disease, in reaction to therapeutic treatments,
under diverse environmental conditions, and in a multitude of other
experimental designs. RNA-Seq empowers scientists to identify both recognized
and new features in a single test. This includes the detection of transcript
isoforms, gene fusions, single nucleotide variants, and other elements, all
without the constraint of pre-existing knowledge.
 Introduction to RNAseq pipeline
1. Quality Control (QC)
•Purpose: Assess the quality of the raw sequencing reads to identify potential issues such as
low-quality bases, adapter contamination, or duplicated sequences.
•Tools: FastQC, MultiQC
•Why: Ensuring data quality at the beginning prevents erroneous downstream analyses and
saves computational resources.

2. Trimming & Filtering

•Purpose: Remove low-quality bases and adapter sequences from the reads.
•Tools: Trim Galore!, Trimmomatic, Cutadapt

Why: Low-quality sequences and adapter contamination can introduce bias and inaccuracies
in downstream analysis, such as alignment.
3. Read Alignment
•Purpose: Map the quality-controlled reads to a reference genome or transcriptome.
•Tools: STAR,HISAT2, Bowtie2

Why: Mapping reads to a reference genome is necessary to identify which genes are
being expressed and how abundantly.

The types of RNA-seq reads (adaption of the Figure 1a from

Kim et al. 2015): reads that mapped entirely within an exon
(in red), reads spanning over 2 exons (in blue), read
spanning over more than 2 exons (in purple)
 Principle of spliced mappers:
(1) identification of the reads
spanning a single exon, (2)
identification of the splicing
junctions on the unmapped
reads
4. Post-alignment QC
•Purpose: Assess the quality of the aligned reads.
•Tools: Samtools, Qualimap
•Why: To ensure that the majority of the reads align to the reference genome properly,
which is crucial for accurate quantification.

5. Transcript Assembly & Quantification

•Purpose: Quantify gene or transcript expression levels from the aligned reads.
•Tools: StringTie, Salmon, Kallisto, FeatureCounts
•Why: The aim is to calculate the abundance of transcripts, which will be used for
downstream analysis like differential expression.

6. Differential Expression Analysis

•Purpose: Identify genes that are differentially expressed between conditions (e.g., control
vs. treated).
•Tools: DESeq2, edgeR, limma

Why: Differential expression analysis helps uncover how experimental conditions affect
gene expression.
7. Functional Enrichment Analysis
•Purpose: Determine the biological significance of the differentially expressed genes
(DEGs).
•Tools: DAVID, GSEA, ClusterProfiler
•Why: Enrichment analysis reveals pathways or processes that may be regulated
under the experimental conditions.

8. Visualization
•Purpose: Create visual representations of the data, such as heatmaps, volcano plots,
and PCA plots.
•Tools: ggplot2, pheatmap, ComplexHeatmap
•Why: Visualization helps interpret and communicate the results clearly.

9. Validation
•Purpose: Confirm the differential expression results using a complementary method.
•Tools: qPCR, Western blot
•Why: Validation ensures that the RNA-Seq findings are accurate and biologically
relevant.
Lab Exercise II RNA-seq data analysis - Part one
Materials:
Galaxy: https://usegalaxy.org/
Dataset :https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241397
Paper:https://onlinelibrary.wiley.com/doi/10.1002/ece3.10976
Assignment part one word file (download from the blackboard)

Methods:
1.Click on the link Dataset and read the research description and click on the
link paper, to find the diet section in the Methodology, answer the Q1 in
data analysis assignment.
2.Click on the SRA Run Selector, it will jump to the dataset download page,
read the information about dietary condition and answer the Q2 in data
analysis assignment
3. In the SRA Run Selector page, click on Accession list in the
select section, it will download the list of all accession numbers.

4. Click on Galaxy: https://usegalaxy.org/ and sign up an

account and log in.
5.Click on the History + icon to create a new history and
rename it as RNAseq.
6. Click on the Upload and Choose local file, upload the
accession list just downloaded.
7. Once complete uploading, In the Tool section, click on the
Get data, choose Download and Extract Reads in FASTQ
8. In the tool parameters section, under select input type, choose List of SRA
accession, one per line and make sure the uploaded accession list is chosen.
Select output format as gzip compressed fastq. Enable the Email notification and
click on Run tool. It takes about 3 hours to complete download process.
 9. In the right side under the RNAseq history, you will see the Single-end data and pair-end
data. Delete the Single-end data collection as it is empty and then search for tool Flatten
collection, choose pair-end data as input collection. Click on Run Tool, this takes 20 seconds.

10. In the Tool section, search for FASTQC, click on it and under the Tool Parameters, “Raw
read data from your current history” click on Dataset collection and choose: Output of
Flatten collection. Keep the rest setting as dafault and Run Tool. This takes about 10mins.
11. Inspect the webpage output of FastQC tool for the SRR25724539 sample (forward and reverse),
answer the Q3 in the assignment.
12. As it is tedious to inspect all these reports individually, we will combine them with tool MultiQC ,
parameters as follows:
13. Under the History, to inspect the MultiQC webpage
result, click on the eye icon. Inspect the result and answer
the Q4 in the assignment.
14. Use Trim Galore! tool to remove the adaptors, the parameters are
as below picture EXCEPT Adapter sequence to be trimmed choose
the one you answered in Q3. Run tool and it takes about 20 mins.
15. Use Flatten collection to flatten the collocation of Trimmed
reads, then use the FastQC to check the quality for post-trimmed
reads, lastly use MultiQC to inspect the result of the adapter
content again, and answer the Q5 in the assignment.

16. Download the Ensembl gene annotation for Drosophila

melanogaster by click on this link:https
://zenodo.org/record/6457007/files/Drosophila_melanogaster.BDG
P6.32.109_UCSC.gtf.gz
17. Upload this file to Galaxy and verify that the datatype is
gtf, and that the database is dm6. Click on Save to upload.
18. Use RNA STAR with the following parameters to map
your reads on the reference genome: (This process takes
about 2 hours 40 mins)
•“Single-end or paired-end reads”: Paired-end (as collection)
•param-collection “RNA-Seq FASTQ/FASTA paired reads”: the Trim Galore!
on collection N: Reads (output of Trim Galore! tool)
•“Custom or built-in reference genome”: Use a built-in index
•“Reference genome with or without an annotation”: use genome reference
without builtin gene-model but provide a gtf
•“Select reference genome”: Fly (Drosophila melanogaster): dm6 Full
•param-file “Gene model (gff3,gtf) file for splice junctions”:the imported
Drosophila_melanogaster.BDGP6.32.109_UCSC.gtf.gz
•“Length of the genomic sequence around annotated junctions”: 36
•“Per gene/transcript output”: Per gene read counts (GeneCounts)
•“Compute coverage”: Yes in bedgraph format
19. Use MultiQC to aggregate the STAR logs with the
following parameters and answer the Q6 in the
assignment.
20. Inspect the counts from any sample in the collection RNA
STAR on collection N: reads per gene, and answer the Q7 in the
assignment.
21. Use Select last from a dataset to remove the first 4 lines with
the following parameters:
22. Cut columns from a table with the following
parameters:
23.Rename the collection as FeatureCount-like files
24.Use the tool Gene length and GC content, to extract the gene
lengths for each gene, parameters are used as follows:
25. Use tool Sort ,to sort the gene with descending order,
inspect the result and answer the Q8 in the
assignment. Parameters are used as follows:
Assignment
Assignment 8 (2%)
1.Complete the Microarray simulation:
https://learn.genetics.utah.edu/content/labs/microarray
2.Answer the questions below:
3.What’s purpose of using the green and red labeling mix?
4.In the result, what does green, red, yellow and black mean?

Data analysis assignment part 1/2 (5%)

5.Download the data analysis assignment part one word file.
6.Complete the questions and submit it before next class starts.