LAB 2: GENE EXPRESSION IN ARABIDOPSIS

GENETIC ENGINEERING

MARÍA LUZ ANNACONDIA

maria.luz.annacondia@slu.se
Contents
Lab practical schedule:.................................................................................................................................. 2
Reports deadlines: ........................................................................................................................................ 2
REVERSE TRANSCRIPTION-QUANTITATIVE PCR (RT-qPCR) ........................................................................... 4
EXPERIMENTAL DESIGN ................................................................................................................................ 6
1 SAMPLE PREPARATION ......................................................................................................................... 7
1.1 STEPS ............................................................................................................................................. 7
2 RNA EXTRACTION AND DNase I TREATMENT ....................................................................................... 8
2.1 STEPS ............................................................................................................................................. 8
3 cDNA SYNTHESIS ................................................................................................................................. 10
3.1 STEPS ........................................................................................................................................... 10
4 qPCR SET-UP........................................................................................................................................ 12
5 ANALYSIS OF THE RESULTS.................................................................................................................. 13
5.1 STEPS ........................................................................................................................................... 13
6 INSTRUCTIONS FOR WRITING THE REPORT ........................................................................................ 14

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Lab practical schedule:

Introduction Thursday 16.11.17 – 10.00 to 12.00 h

Step 1: Sample preparation Friday 17.11.17 – 09.00 to 12.00 h

Step 2: RNA extraction and DNase I Friday 17.11.17 – 09.00 to 12.00 h

Step 3: cDNA preparation Monday 20.11.17 – 09.00 to 12.00 h

Step 4: qPCR set-up Thursday 23.11.17 – 09.00 to 12.00 h

Step 5: Results analysis Thursday 23.11.17 – 14.00 to 16.00 h

Extra time Friday 24.11.17 – 10.00 to 12.00 h

Discussion of the results Wednesday 29.11.17 – 10.00 to 12.00 h

DAY 1 DAY 3

DAY 4

DAY 2
Reports deadlines:

First deadline Wednesday 13.12.17 – 23.59 h

Final deadline Friday 12.01.18 – 23.59 h

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SAFETY NOTES
 Work in fume hood when using ß-mercaptoethanol.
 Use lab coat with long sleeves and gloves.
 Use safety glasses and thick gloves when handling liquid nitrogen.
 Wear nitrile gloves when handling ethidium bromide.

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REVERSE TRANSCRIPTION-QUANTITATIVE PCR (RT-qPCR)

The RT-qPCR is a technique based on two steps, a reverse transcription followed by a quantitative PCR.
The reverse transcription consists of the generation of complementary DNA (cDNA) using RNA as the
template and the action of an enzyme called reverse transcriptase. This enzyme, just as the DNA
polymerases, needs a primer to be able to start synthetizing the new DNA strand. Usually, cDNA
synthesis kits come with the different primer option, random primers, which are usually hexamers with
different sequences that can bind randomly to the template, and oligo(dT) primers, which bind to the
poly(A) tale of the mRNAs. Once the cDNA is obtained, it is used as the template for the qPCR.
qPCR, just as the PCR, is based on an enzyme called Polymerase, but in this case the detection of the
synthetized amplicons is achieved by using fluorescence and a special thermocycler, capable of
detecting this fluorescence. So, in this technique the PCR product is marked with a fluorophore, a
molecule which binds to the DNA and emits light when it is light-excited. In this way, the amount of
fluorescence which is measured in each cycle is directly proportional to the amount of synthetized
product and it increases in an exponential way, just like a regular PCR.
However, it takes some cycles to detect the fluorescence, because at the beginning of the process the
amount of product is not enough to produce the minimum amount of fluorescence that the
thermocycler is capable of detecting. These minimum level of fluorescence marks the threshold, which is
important because the interesting value is the cycle in which this threshold is overtaken, named the Ct
value.

As the initial amount of cDNA template for each studied gene is not the same, the results from the qPCR
need to be normalized using housekeeping genes to be able to compare them. A housekeeping gene is a
gene which is constitutive expressed, normally they are genes that are needed for the basic
maintenance of cellular functions and are expressed in the same levels in normal and non-normal
(pathological conditions, stress, etc.). Some examples are ubiquitin, actin or GADPH (Glyceraldehyde 3-
phosphate dehydrogenase).
Finally, to know if the primers are working properly and they are not forming primer dimers, a melting
curve is usually performed. In this process, after the quantitative PCR is finished, the temperature is
progressively increased and the fluorescence is again measured. If the primers are working as expected,
the melting curve would have a unique pick, which is the temperature in which the primers and the
amplicon are denaturalized and they stop realizing fluorescence. However, if the primers are forming

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dimers, the melting curve would have to picks, the first one represents the primer dimers and the
second one the primer-amplicon product.

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EXPERIMENTAL DESIGN

Our experimental design consists of 6 different Arabidopsis thaliana genotypes, the wild type (in this
case the accession Col-0) and 5 different mutants (named ddm1, drm2, cmt3, ago4 and ddc), whose
background is Col-0. The following table contains a summary of information about these mutants:
Epigenetic
Mutant Full name Locus
mechanism
Cytosine
ddm1 DECREASED DNA METHYLATION 1 AT5G66750
methylation
DOMAINS REARRENGED
drm2 AT5G14620 CHH de novo
METHYLTRANSFERASE 2
cmt3 CHROMOMETHYLASE 3 AT1G69770 CHG methylation
ago4 ARGONAUTE 4 AT2G27040 DNA methylation
AT5G15380,
ddc Triple mutant drm1 drm2 cmt3 AT5G14620, DNA methylation
AT1G69770

All these genes are involved in the regulation of gene expression through epigenetics. You can read
more information about the role of these genes in the Arabidopsis Information Resource (TAIR)
(https://www.arabidopsis.org/).
For the purpose of this experiment (and in general in molecular biology) we work with 3 individual tissue
samples. Each of these samples is a pool of 4 different biological samples, this means we are going to
mix the tissue from 4 different plants to make one biological sample. By doing this, we are going to have
more plant material (in case we need to repeat the experiment or do additional confirmations of the
result) and at the same time we are going to minimize the possible variability among plants. So, we are
going to have 3 different samples for each genotype which are made from 4 different plants, which
means that in total we are going to have 18 samples.
We are going to analyze the expression of two genes, ALTERED MERISTEM PROGRAM 1 (AMP1 which its
AGI code is AT3G54720) and a gene that is located in its 5’ region, AT3G54730 (which its function is
unknown yet, that is the reason that it does not have a name), in Arabidopsis leaves. As a reference for
gene expression we will use a housekeeping gene (a gene that has the same expression in all mutants
because it has a structural role in the cell) UBIQUITIN10 (AT4G05320).

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1 SAMPLE PREPARATION

We are going to work with Arabidopsis thaliana leaves. Each person is going to prepare 3 different
samples, in total, we will have 18 samples (6 different genotypes x 3 biological replicates).
1.1 STEPS
1. Prepare 3 1.5 ml tubes with glass beads. Don’t forget to properly label them.
2. Take the leave from each plant in the pot and put them in one of the labeled tubes. In this way, you
will have 3 pools made with 4 leaves each one.
* Note: from this moment on, you have to work fast in order to prevent that the samples thaw. If this
happens the RNA will degrade, compromising the quantitative PCR reliability.
3. Transfer the tube to the liquid nitrogen container as quick as possible. We sure that the tubes are
correctly closed. And be extremely careful while working with liquid nitrogen!
4. Grind the samples using a dental mixer. 10 seconds of grinding should be enough!
5. Put the grinded samples back in the liquid nitrogen container.

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2 RNA EXTRACTION AND DNase I TREATMENT

There are many different RNA extraction protocols in molecular biology,

which have to deal with the specific characteristics of RNA. First, RNA can
be degraded very easily due to its own unstable structure and by the
ubiquitous presence of enzymes that degrade RNA and are naturally
present in the cell or the surface of the tissues (where they degrade RNA
pathogens like viruses). Most RNA extraction methods also use the
hydrophilic nature of the RNA molecule in order to separate nucleic acids
from proteins. We are going to use the RNeasy Mini Kit of Qiagen to extract
the RNA, a commercial method based on the selective capture of RNA to a
membrane placed inside of a centrifuge tube.
2.1 STEPS
1. Prepare enough β-mercaptoethanol +RLT buffer for your samples (N) +1
extra. Each sample will have 450 μl β+RLT. To make β+RLT (in the fume
hood) add 10 μl β-mercaptoetanol per 1 ml RLT buffer (RNeasy kit) and mix
well.
2. Add 450 μl β+RLT to each tube containing the ground samples and mix
gently.
3. Transfer the lysate to a spin column (lilac) placed in a 2 ml collection
tube, and centrifuge for 2 min at full speed.
4. Carefully transfer the supernatant of the flow-through to a new
microcentrifuge tube without disturbing the cell-debris pellet in the
collection tube. Use only this supernatant in subsequent steps.
5. Add 0.5 volume of ethanol (96-100%) to the cleared lysate, and mix
immediately by pipetting. Do not centrifuge.
6. Transfer the sample (usually 650 µl), including any precipitate that may
have formed, to an RNeasy spin column (pink) placed in a 2 ml collection tube. Close the lid gently, and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.
7. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at
≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.
8. Add 10 µl DNase I stock solution to 70 µl Buffer RDD. Mix by gently inverting the tube, and centrifuge
briefly to collect residual liquid from the sides of the tube.
9. Add the DNase I incubation mix (80 µl) directly to the RNeasy spin column membrane, and place on
the benchtop (20-30 °C) for 15 min.
10. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at
≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.
11. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at
≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.
12. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at
≥8000 x g (≥10,000 rpm) to wash the spin column membrane.

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13. Place the RNeasy spin column in a new 1.5 ml tube and add 30 ul RNase-free water directly to the
spin column membrane. Close the lid gently, and centrifuge 1 min at ≥8000 x g (≥10,000 rpm) to elute
the RNA.

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3 cDNA SYNTHESIS

Before preparing the cDNA synthesis reaction, we need to check the quality and concentration of our
RNA. For that, we are going to prepare an agarose gel to visually check the lack of degradation of the
most notorious RNA bands (ribosomal RNA, which can make up to 75% of the total RNA in the cell) and
we are going to measure the concentration of RNA in our liquid solution through spectrophotometry by
means of a Nanodrop. With this two quality checks we will know the concentration of RNA in our
samples (spectrophotometry) and also if that RNA is not degraded (gel visualization).
3.1 STEPS
1. Prepare 50 ml of a 1% agarose gel. For this, dissolve the appropriate amount of agarose in 1x TBE
buffer by using a microwave.
2. Once dissolved, let the agarose mixture cool down, more or less until you can hold it.
3. Add 2.5 µl of Ethidium Bromide to the agarose mixture and mix well.
4. Pour the mixture in the tray.
5. While the gel is polymerizing, prepare you the samples that you are going to load. For this, mix 2 µl of
your RNA, 3 µl of water and 1 µl of 6x loading buffer.
6. Once the gel is ready, load your samples and run it at 120 V during aprox. 20 min. After this time,
analyze it under the UV light.
7. While the gel is running, we are going to check the RNA concentration using a Nanodrop.
8. Calculate the RNA volume you need in order to have the appropriate amount of initial RNA template
for the cDNA synthesis (which will be determinate in function of the obtained total RNA).
* NOTE: This initial RNA template volume cannot be more than 11 µl because the total volume of the
reaction is 20 µl and we need to add more components.
9. The cDNA synthesis will be carried out in a PCR machine, so prepare the PCR tubes that you need.
10. Mix the appropriate amount of RNA with 1 µl oligo (dT)18 and fill up to a final volume of 12 µl with
water if it is necessary. Mix well and centrifuge briefly.
11. Place the tubes in a thermocyler for 5 min at 65 °C.
12. While the tubes are in the thermocyler, prepare the necessary amount of mastermix using the
following recipe (and adding one extra sample to avoid running out of it). Don’t forget that you will need
3 more wells per primer for the negative control (water):
1 sample Your mastermix
5x Reaction Buffer 4 µl µl
RiboLock RNase Inhibitor (20 U/µl) 1 µl µl
10 mM dNTP Mix 2 µl µl
RevertAid M-MuLV RT (200 U/µl) 1 µl µl
Total volume 20 µl µl

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13. Add the appropriate amount of mastermix in each PCR tube. Mix well and centrifuge briefly.
14. Place the tubes in the thermocycler and create the following program: 60 min 42 °C; 5 min 70 °C and
hold 8 °C.

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4 qPCR SET-UP

1. Design the qPCR plate, try to make it as easy to pipet as possible. You will need 3 analytic replicates
for each biological replicate, which means that each sample will take 3 wells. Don’t forget the 3 extra
wells you need for the negative controls of each master mix.
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H

2. It is not possible to accurately measure cDNA concentration, thus we have to assume that the RNA to
cDNA conversion was 100% efficient. For the qPCR reaction 40 ng of cDNA will be used in a 20 uL
reaction. To make pipetting easier and for accuracy purposes it is recommended to dilute the cDNA.
Dilute the cDNA to 10 ng/ul.
3. Prepare the 3 different master mixes, each one for one primer, using the following recipe:
1 Your master mix Your master mix Your master mix
sample (Primer 1) (Primer 2) (Primer 3)
5x HOT FIREPol EvaGreen
4.0 µl µl µl µl
qPCR mix
Primers F+R 0.8 µl µl µl µl
DNA template 4.0 µl µl µl µl
Water 11.2 µl µl µl µl
Total volume 20 µl µl µl µl

* NOTE: Make sure that you add the correct primer to each master mix!
4. Pipette the master mixes and the samples in the correct well. Seal the plate and centrifuge.
5. Run the RT-qPCR.

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5 ANALYSIS OF THE RESULTS

5.1 STEPS
You can use Excel to analyze your results and obtain the appropriate graphs.
1. Calculate the average value of the Ct from the three analytical replicates for each biological sample.
2. Calculate the relative expression of the studied gene normalized with the housekeeping gene by
using the formula 2^-(CtGENE-CtHK), where A is the Ct of the studied gene and B the Ct of the housekeeping
gene.
3. Perform a t test using the Excel function t.test comparing the Cts from the mutants with the Cts of the
wild type to see if the differences between them are statistically significant.
4. Calculate the standard deviation of the normalized expression.
5. Normalized the standard deviation by dividing it by the sum of the normalized expression of the gene
in the wild type.
6. Calculate the relative expression of the gene in the mutant compare to the wild type by dividing the
sum of the normalized expression of the gene in the mutant by the sum of the normalized expression of
the gene in the wild type.
7. Plot the relative expression normalized with the wild type in a histogram. Use the normalized
standard deviation as the error bars.
At the end of the result analysis you should have a histogram that looks like this one:

1.4
1.2
1
0.8
Gene1
0.6
Gene2
0.4
0.2
0
wt mut1 mut2

Also, you should provide a table with the final results. For example:
Gene1 Gene2
wt mut1 mut2 wt mut1 mut2
Relative expression 1 1.006758 0.766021 1 0.887555 0.658125
Normalized stdev 0.011159 0.021573 0.032865 0.279102 0.143083 0.01683
t-test 0.880168 0.017658 0.846129 0.519182

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6 INSTRUCTIONS FOR WRITING THE REPORT

The report should be in the form of a scientific article, and it should summarize both your literature
survey as well as your lab work. It should be written in English. Look in established international
scientific journals to see how a research article is written and how to cite. There are also many articles
and books about how to write scientific articles, see for example Frey (2003), the concise guideline in Sci
Dev Net’s “How to write a scientific paper” and “Presenting Science”, from the Biology Education
Centre. Remember how to write names of proteins (ABI1), genes (ABI1) and mutants (abi1) in
Arabidopsis!
To be able to write the report you need to read scientific articles and/or books about the subject. Note
that you may not cite unconfirmed web pages.
The report should be written in a focused and condensed manner. Therefore there is a strict limitations
on how many words it can be. The Introduction + Discussion can together be max 1500 words. The
Materials and Methods and the Results section are not limited but should be condensed and succinct.
- Title: Try to find a title that includes (the most important) results of your experiments. Don’t
forget to write your own name on the front page of the report!
- Abstract: Short summary of the entire report, including the rationale, methods, key results and
the main conclusion, including key points of discussion. Max 250 words.
- Introduction: Include the theoretical background and a survey of the pertinent literature leading
up to the point where your work is of importance, i.e. the “gap-in-knowledge” in current
literature. You should take care to have a focused and to-the-point introduction, and essentially
introduce only information that is of need for the reader to understand your results and
discussions. You should state clearly the scope and objectives of the study (which is to fill this
gap, and how). The introduction can end with a brief summary of principal findings showing
how/if you closed the gap. Do not forget references and how to write them! Max 750 words.
- Materials and methods: The main purpose of this section is to give enough information such
that all experiments presented can be repeated by someone else. You can and should refer to
manuals of the kits that were used, but only if you followed these protocols exactly, otherwise
any modification should be specified. The methods should be presented in a running text, and
follow a chronological order. Information about every step of the experiment should be
provided, starting from sample preparation and ending in the analysis of results. This text does
not contain any theory or results only the methods and the materials you used. Should be
written in third person, i.e. do not use ‘I’ or ‘we’. See published reports for how to write
materials and methods.
- Results: You must have a running text describing what you did and your results. Note that you
must refer to all pictures, photos and tables in the running text, and that figures should appear
in the order they are mentioned in the text. Remember to make figure legends to your pictures,
photos and tables. The reader should neither have to look at the figures/tables to follow your
arguments nor should the reader have to search the running text to understand figures/tables.
Figure legends to pictures and photos are written below the figure, and table legends are
written above the table. Remember that negative results are also results. You should select the
results you think are crucial for answering your initial questions. Additional results that is
important, but not absolutely essential to prove your point should be presented in the
Supplementary Data section.
- Discussion: In the discussion the main focus should be to follow up what you started in the
Introduction, and discuss how your results fit in the context of what is previously known. You

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 can also compare results of different methods/materials. Major error sources can be included
only if you are convinced that they affected the outcome of the experiment –if you discuss a
source of error, focus on what you can do to improve the experiment. If you choose to include
error sources, you should be able to explain how these affected the result of your experiment. A
section on error sources should not include more than a few sentences. It should NOT make up
your entire discussion. You can conclude the discussion by proposing hypotheses and models
based on your results, but try not to be too speculative. Max 750 words.
- References: Whenever you write about information published in another paper, you must
acknowledge the source immediately and give the reference. If two authors are involved,
include both surnames in this reference. However if more authors are involved, you may use 'et
al', an abbreviation of Latin meaning 'and others'.” A list of references ordered alphabetically by
author's surname must be provided at the end of your paper. The reference list should contain
all references cited in the text and no other. Include details of the authors, year of publication,
title of article, name of journal or book and place of publication of books, volume and page
numbers.
Please know that you cannot copy sections of text - this is plagiarism and is taken very seriously.
References:
Frey, P.A. (2003) Guidelines for writing research papers. Biochemistry and Molecular Biology Education
31(4): 237-241
http://www.scidev.net/global/publishing/practical-guide/how-do-i-write-a-scientific-paper-.html
Presenting Science, Biology Education Centre, 2011.

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