Comparison of the expression of

Ribonucleotide Reductases grown in

sputum of a patient with Cystic
Fibrosis and in a laboratory strain

Authors:
Ferran Clot Razquin
Elisabeth Sánchez Jiménez
Elsa Usán García
Mentor: Judith Trepat
Jesuïtes-el Clot 2nd Grade GSFP Laboratory Technician 2017-2019
P9. MACROproject April 2019 | Volume 1 | Article 1

ABSTRACT

The purpose of our study was to compare the expression of the genes generated by the RNR of a
patient with CF and those generated by PA14. It would allow study the behaviour and expression of
the genes to compare how new antibiotics affected them. So, the whole purpose of these studies, is
to create an antibiotic able to break this biofilm and directly attack the Pseudomonas. To carry out
this study we used two main techniques, qRT-PCR and Western Blot. The results obtained were
negative, showing an absence of Pseudomonas in the sample of the patient. Subsequent confirmatory
tests of the presence of P. aeruginosa in the sample of the patient were done. These were confusing
results, that although the confirmatory test showed a positive result indicating presence of
Pseudomona, negative results in the experiments (WB and PCR) showed failure to compare the
expression of the genes from the patient with those from the strain PA14. On the other hand, the
process following the steps of protocols was performed correctly, because every test done during the
procedures showed right results. So, at that point, the different hypothesis raised were the following:
there was not enough Pseudomona in the patient lungs and we must had carried out the whole
experiment with a colony grown in the first plating, so we make sure we are working with P.
aeruginosa.

KEYWORDS: Cystic Fibrosis, RNR, CFTR, Pseudomonas aeruginosa, Western Blot, PCR
TABLE OF CONTENTS

INTRODUCTION .............................................................................................................. 2
MATERIAL....................................................................................................................... 4
METHODS ........................................................................................................................ 5
RESULTS .......................................................................................................................... 9
DISCUSSION .................................................................................................................. 12
CONCLUSIONS .............................................................................................................. 14
ACKNOWLEDGEMENTS .............................................................................................. 15
REFERENCES ................................................................................................................ 15
ANNEX ........................................................................................................................... 16
P9. MACROproject April 2019 | Volume 1 | Article 1

INTRODUCTION
Cystic fibrosis (CF) is a rare chronic and progressive genetic disorder that affects more than 70,000
people around the world, in white populations the pathology occurs in 1 of 3000 births, characterized
by dysfunctional secretory epithelial cells that cause obstructions in the respiratory tract and pancreatic
behaviours. (Sanz DJ, 2017)
Is caused by a mutation in a gene that encodes cystic fibrosis transmembrane conductance regulator
(CFTR) protein, which is expressed in many epithelial cells and blood cells. (O'Sullivan BP F. S., 2009)

Although CFTR functions mainly as a chloride channel, it has many other regulatory roles, including
inhibition of sodium transport through the epithelial sodium channel, regulation of the outwardly
rectifying chloride channel, regulation of ATP channels, regulation of intracellular vesicle transport,
acidification of intracellular organelles, and inhibition of endogenous calcium-activated chloride
channels. (A., 2005)
CFTR is also involved in bicarbonate-chloride exchange. A deficiency in bicarbonate secretion leads to
poor solubility and aggregation of luminal mucins. (PM., 2008)
Also, CF can affect a wide number of organs, but basically it endangers lungs and the digestive system,
this last one due to the pancreatic behaviours. While the patient ages and the disease progresses, it
makes other important organs stop functioning efficiently. (O'Sullivan BP F. S., 2009) Annex I

The treatment of Cystic Fibrosis is based on three fundamental pillars: getting an adequate nutrition
(the antibiotic therapy used at a gastrointestinal level to improve the adequate nutrition in patients with
CF consists in specific vitamins, enzymes and minerals), using drugs that fight against respiratory
infection and inflammation (together with nebulized antibiotics and saline), and performing, regularly,
physical therapy consisting of respiratory physiotherapy, strengthening exercises of the thorax
musculature to prevent deformities, and the practice of some sport. (Quística, 2019)

The bacteria that causes the infection forms a membrane that we call 'biofilm' in which the optimal
conditions are created for it to develop and reproduce.
Pseudomonas aeruginosa, a Gram-negative bacterium, is characterized by its versatility that enables
persistent survival under adverse conditions. It can grow on very different energy sources and acquire
resistance to antimicrobial agents. As a strict aerobe, P. aeruginosa can grow by anaerobic nitrate
(NO3−) respiration when present. (Masanori Toyofuku, 2018)
It is one of the worst bacteria that can be found in respiratory pathologies. It is the cause of the disease
symptoms worsening in patients with CF. This bacterium, once it colonizes the individual, is very
difficult to make it disappear. (Fleming, 2016)

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When the P. aeruginosa (PA) bacteria causes chronic lung infections, it means that it has been able to
form a mature biofilm in situ that allows it to grow and adapt. (IBEC, 2019)
The biofilm creates ideal conditions for the bacteria, facilitates communication between cells and,
consequently, the infection increases, developing future resistance to antibiotic treatment and worse
response to the immune system of the individual, when reducing the concentration of oxygen inside the
biofilm. (Fleming, 2016)

P. aeruginosa is one of the few bacteria that codes for the three classes of Ribonucleotide reductase
(RNR) known in its genome: Class I, which is oxygen dependent; Class II, which is oxygen
independent; and Class III, which is sensitive to oxygen and can only function under strict aerobic
conditions, except when there is presence of nitrates. For our study, we focused on type I and II RNRs,
which is where P. aeruginosa expresses the nrdA and nrdJA genes necessary for the development of
our experiment.
Ribonucleotide Reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides
(DNA precursors) for the synthesis of DNA in each living cell. This enzyme is responsible for
converting ribonucleotides into deoxyribonucleotides.
They have contributed to the emerging of the genetic material that exists today. Strict control of the
RNR activity and the size of the dNTP aggrupation is important, since group disturbances increases
mutation rates, replication anomalies, and genome instability.
They are essential enzymes for all organisms, since these proteins provide the dNTP necessary for the
replication of chromosomes and the repair of DNA damage. The contribution of RNR to these key
reactions makes this enzyme a perfect target for the design of compounds that inhibit cell growth in
cells with altered cycles (cancer cells) or during viral, bacterial or protozoal infections. (Torrents, 2014)
These enzymes are responsible for increasing the capacity of PA to grow in the different aerobic and
anaerobic environments generated along biofilms. (IBEC, 2019)

RNRs are essential for the cell division of the strain PAO1 (Pseudomonas aeruginosa) that supplies the
necessary monomers for the synthesis and repair of the bacteria's DNA, which allows it to grow and
adapt to diverse environmental conditions, even during infection. The researchers discovered that the
absence of activity of the RNRs leads to cell elongation in the PAO1 strain, indicating lack of growth.
In addition, the genetic expression of the RNR during anaerobiosis -process that occurs in the absence
of oxygen- differs between the clinical strains of P. aeruginosa and those used in the laboratory, since
the Class III RNR - also Class I and Class II but to a lower degree- is much more expressed in clinical
isolates of P. aeruginosa compared to the laboratory strain because they modulate the growth of the
biofilm and therefore the growth and development of the bacteria. (Fleming, 2016)

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The type of RNR class will affect the adaptability of microorganisms because they operate in a range
of different environmental conditions; therefore, bacteria that code for more than one RNR class can
survive in a wide range of ecological niches, such as bacteria P. aeruginosa. (Torrents, 2014)

The main goal of our investigation is to observe and compare the genetic expression of Ribonucleotide
Reductases grown in a laboratory strain (in which we control both the exponential and the stationary
growth) and the ones grown in the lungs of a patient suffering Cystic Fibrosis. This experiment will
allow the study of the expression of these genes in real life, which is going to make possible the
recreation of them in the laboratory through future experiments in a way that will enable the
investigators to try the new medicines and antibiotics with them and observe the effectivity that those
would have in the patients that has been infected by Pseudomonas.
For that, we are going to work hand in hand with phD students at the Institute for Bioengineering of
Catalonia (IBEC) which are already investigating with the group of Dr. Eduard Torrents “Bacterial
Infections and Antimicrobial Therapies”, dedicated to research of new therapies against the bacteria
that attack and infect patients with Cystic Fibrosis.

MATERIAL
Sample of the study
Our sample is sputum from a patient with CF, diagnosed from birth, with the following mutations:
F508del and 1811 + 1.6kb A>G.
The patient is 20 years old. At the time of the experiment, she is not taking any specific treatment.

Reagents and items

For the realization of the experiment we need the following reagents: PBS 1x as a buffer, RNA protect,
sterilized and nuclease-free H2O milli-Q, LB media and Crystallic Violet, PA14 (Pseudomona
aeruginosa) strain prepared for use in laboratories, Sonication Buffer, RNA extraction Kit, RNA
purification Kit, DNA elimination Kit, DNAse buffer and EDTA for the treatment of DNAses, Primers,
DreamTaq MasterMix for the PCR of the DNA absence test, 2% Agarose gel with Ethidium Bromide
and 100kb ladder, dNTP Mix and Maxima Reverse Transcriptase with buffer for the cDNA synthesis,
Bradford reagent for Protein Assay, Laemmli Buffer, Resolving and Stacking gels for Western Blot and
ladder, TGS 1x which is the buffer for the Western Blot electrophoresis, powdered milk to block the
membrane, antibodies anti-A and anti-J and Anti-Rabbit, 0’05% Tween20 as a detergent, Luminol
Enhancer Solution with Peroxide Solution, SYBR 2x MasterMix for the qRT-PCR.

Equipment
The main equipment required for this experiment is: microbiological safety cabinet, homogenizer,
different types of centrifuges, Branson sonictor Annex II, TECAN Microplate Reader, Thermoblock,
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PCR Thermal Cycler, Electrophoresis tanks and machines for DNA absence test and Western Blot
Annex III, Criterion Blotter Turbo™ Transfer System for the membrane, Orbitron, Nanodrop,
StepOnePlus Real Time PCR System, ImageQuant LAS 4000 mini.

METHODS
- Extraction of sputum from patient with CF
- Processing sample homogenizing with PBS buffer (and RNA protect for the sample that will
be used for the qRT-PCR), centrifuge and freeze the pellets Annex IV and V
- Plating sample of sputum in LB Agar, restreak days later some grown colonies in selective
media with Crystallic Violet, and do two Overnights with LB Broth (one with a purple colony
and the other with a green colony) to ensure there are or there aren’t Pseudomonas Annex VI

Western Blot
- Sonicate the pellet resuspended in Sonication buffer. Centrifuge and freeze the supernatant
Annex VII
- Quantify protein with Bradford (Abs. = 595nm) Annex VIII and IX
- Prepare the samples to load the gel with the specific concentration of proteins (given by the last
step), Laemmli buffer and water, and incubate for 10min. at 100ºC in the Thermoblock
- Load and run the gel at 40mA (20mA per gel) during more than an hour Annex X
- Transfer it to the membrane Annex XI
- Block membrane with milk overnight placed on the Orbitron in the 4ºC camera Annex XII
- Immerse the membranes separated in the primary antibodies (Anti-A and Anti-J respectively)
and wash with PBS + Tween during 15min. two consecutive times; all at 4ºC to optimize the
union of the antibodies
- Repeat last step with the secondary antibody (Anti-Rabbit) with both membranes
- Develop the membrane with Luminol solution in the ImageQuant

qRT-PCR
- RNA extraction and purification Kit
- DNA elimination Kit
- DNAse treatment Annex XIII
- DNA absence test throughout a PCR and running an electrophoresis in Agarose gel Annex XIV
- Quantify RNA in Nanodrop
- cDNA synthesis from RNA using SuperScript IV RT, thin the dilution and freeze Annex XV
- Prepare the well plate and set the cycles to start the Quantitative Real Time PCR Annex XVI,
XVII and XVIII
- Analyse plot to set the results
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Statistical Analysis

It has been performed with Excel for the Bradford test of proteins concentration right below to obtain
the pattern line; but we have included in this section also some tables with calculations for the cDNA
and results from the quantification of RNA in Nanodrop and the gel image of the result from the DNA
absence test.

Fig. 1 Resume in Excel of the calculations after the protein quantification with Bradford

Bradford reagent is used to identify proteins in a substance and their concentration in the sample. In our
case, it was used with 5 controls of known concentration of BSA, and with 2 samples: PA14
(Pseudomonas strain), and the sputum from the patient with CF.
This plot about the Bradford test shows the different quantities of each substance (sample, Laemmli
buffer and water) and the total amount of mixture to load the gels. We identify the measurements taken
both for the PA14 and for the sputum of our sample. Finally, we have obtained a chart from the BSA
measurements of each concentration, that is a straight line with an R2=0.9961 (almost 1) which is great.
In the bottom data table, we see the original measures of BSA where can be found 3 replicas and the
final average, used to compare with the current samples.
The average value of the blank was also calculated, with a result of 0.304.

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Fig. 2 Results of the electrophoresis for the DNA absence test

We performed a PCR in order to confirm that the purification of the sample for obtaining RNA was
correct. The purpose of this technique is very simple, if there is PCR and amplification, the purification
has not been optimal since there is DNA. If there is no amplification of DNA, means that the previous
steps have been correctly performed. So, our results are great. The ladder indicates the weight of the
band in the Control +, which is pretty high and indicates the presence of DNA, but in the other tracks
there is no band which means no DNA, and these are the expected results.

Fig. 3 Results of the RNA quantification with the Nanodrop Annex XIX

To quantify the amount of RNA available, we have used Nanodrop, which indicates a low RNA quantity
in our sample, but enough to continue the experiments.

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Fig. 4 Calculations in Excel to prepare the cDNA Synthesis

The synthesis of DNA from an RNA template, via reverse transcription, produces cDNA. Reverse
transcriptases (RTs) use an RNA template and a short primer complementary to the 3' end of the RNA
to direct the synthesis of the first strand cDNA, which can be used directly as a template for the PCR.
This combination of reverse transcription and PCR allows the detection of low abundance RNAs in a
sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes.
In our data table, the blue cells indicate the concentration of protein in each sample. The board on the
bottom left indicates the different quantities of each substance to create a mixture that will break the
secondary structures of cytosines and guanines when incubating. Once this happens, the MaximaRT
with the buffer is added and incubated so the reaction takes place and it builds up the complementary
DNA.
Perform the cDNA is a vital step to carry out the PCR.

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RESULTS

Fig. 5 Results of the Western Blot

Fig. 6 Results of the qRT-PCR

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Fig. 7 Amplification plot of the sputum sample and negative control with each respective gene

Fig. 8 Amplification plot of PA14 strain as a result of the qRT-PCR

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Fig. 9 Overnight of LB with a colony of unknown bacteria, turbid with a green colour characteristic to
the growth of Pseudomonas

Fig. 10 Rose pellet after the centrifugation of the Overnights with the green turbid

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DISCUSSION
The results of the quantitative-Real Time Polymerase Chain Reaction are pretty negatives. What we
had expected to see was an amplification plot similar to Fig. 8, where there is an exponential curve
more or less in the middle of the plot (Cycles between 14 - 28) and a Threshold cutting the middle of
the curve. What we get in the amplification plot of our sample (Fig. 7) is a curve that starts almost at
number 35, that the program already perceives it as a negative, which means there is no DNA expressing
the genes (nrdA, nrdJa, nrdD and nrdR). Also, the housekeeping gene (gap A) that we use as an internal
control fails, and the nor C, which shows us the presence of anaerobiosis, doesn’t seem to be right either.

In addition, as we can observe in the results of the Western Blot membrane development, in the
membrane on the left we see the first track with ladder, the second track apparently empty, and the third
one shows the column of PA14 with expression at about 80kDa, which means the expression of gene
NrdJa, but it expose the absence of this gene in our sample. Same happens in the membrane on the right:
it shows expression at about 107kDa which means expression of the gene NrdA, but our sample lacks
it. (Torrents, 2014)

So basically, the results have been not what we expected, that proteins or DNA with this genes
characteristic from Pseudomonas would come out in our sample, so we could compare the proteins
generated by the RNR enzyme inside the patient with CF conditions and those generated inside the
PA14 strain. So, this allow us to think that, fortunately, the patient with Cystic Fibrosis is not infected
by Pseudomonas aeruginosa. But her last results didn’t say the same. (IBEC, 2019)

On the other hand, we carried out a plating on LB agar of the sputum sample from our patient with CF
at the beginning of this experiment, after homogenizing the sample, and grew green-blue bacteria that
we could not recognize exactly what it was; so we decided to do a restreak of some colonies, but in a
selective media for Pseudomonas: Crystallic Violet. We wanted to ensure there was no growth of this
bacteria, no presence of it. Surprisingly, the results looked positive because something grew, but the
colonies were mostly purple and some green-yellow. Therefore, there was presence of it in the original
sample, but to make sure it was Pseudomonas, we did two tubes overnight with LB broth, one with a
green colony and the other one with a purple colony. The next day we observed turbidity in the media,
which meant growth, and after do a centrifugation, the pellet was rose which clearly the bacteria were
Pseudomonas. (Adriana Callicó, 2004)

The purpose of this study was, at least, observe the expression of the genes in our sample of a patient
with FQ, and then, compare it with the expression generated by a laboratory strain; so it has not been
achieved since we had no expression in the different experiments executed, therefore we were not able

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to compare the proteins generated by the Ribonucleotide Reductases of a regular cell line (PA14 strain)
and the ones generated by RNRs inside the patient. (Torrents, 2014)

The main limitation of our study has been the time. We have had very limited time to carry out all the
experiments.
Our knowledges on the subject also affect our efficiency, as in the previous aspect, we have had little
time to train on the Cystic Fibrosis and the RNR enzymes.
Also another limitation was that only one of the members had access to the laboratory, so she was the
only one carrying out all the experiments (helped by the colleagues from the laboratory) and afterwards,
she had to transmit everything she did to the others, which has slowed down the efficiency even more.

We would like to carry out a new experiment with more time, but with the same theoretical base. We
believe the steps we have taken are right, but maybe we could have executed all the experiments with
a colony from the sputum sample grown in LB agar to ensure there were Pseudomonas. Or probably at
some point we missed something, or we failed doing some of the procedures.
So, if it is possible in the future, we would like to improve the experiment, with a larger frame of time,
with a sample from the same patient, and with the help of experts in the field as the ones who helped
carry out the experiment this time. At least to make sure the protocols are correct and give us the
possibility to observe expression and be able to compare the samples and to study the results. (Torrents,
2014)

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CONCLUSIONS
Cystic Fibrosis is a genetic rare disease, that affects people around the world, with prevalence in white
populations, from European descendancy. It is caused by a mutation in a gene that encodes cystic
fibrosis transmembrane conductance regulator (CFTR) protein, which is expressed in many epithelial
cells and blood cells.

The main goal of our investigation was to discover which differences can be found in the genetic
expression of the Ribonucleotide reductases (RNR) from a regular cell strain for laboratories and in a
sample provided by a patient suffering CF.

To develop this study, we have used two main techniques, a quantification Real Time-PCR and a
Western Blot. We also carried out a plating, restreak and overnight of the sputum sample after the results
as a complemented confirmatory test.

We have obtained confusing results, that although the confirmatory test shows a positive result
indicating presence of Pseudomona, negative results in the experiments (WB and PCR) show failure to
compare the expression of the genes of our patient with those of the strain PA14. On the other hand, the
process following the steps of protocols was performed correctly, because every test done during the
procedures showed right results (DNA absence test, RNA quantification with the Nanodrop, Protein
quantification with Bradford, etc.).

So at that point the different hypothesis raised are the following: there was not enough Pseudomona in
the patient lungs -which we are glad to know and happy to notice her about it- and we must had carried
out the whole experiment with a colony grown in the first plating so we make sure we are working with
P. aeruginosa.
We have had very limited time to carry out the experiments and our knowledge also affected our
efficiency, so we haven’t been able to repeat the experiment reproducing the first hypothesis.

Our hope is to repeat the experiment with a larger frame of time, so that we can make it with the same
theoretical base and procedures but with the colony grown in LB agar instead of immediately using
sample homogenized, and get the results expected with genetic expression of RNRs, so it will make
possible to our colleagues of the laboratory to compare this one with that of PA14 or PAO1 and be able
to study the behaviour and expression of the genes to compare how new antibiotics would affect them.
So, the whole purpose of it is to create and antibiotic able to break this biofilm and directly attack the
Pseudomonas. But for that, more studies and tries along the time are required until we manage to make
it to that point.

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ACKNOWLEDGEMENTS
The work was supported in part through grants from the Generalitat de Catalunya, in part from the
Associació Catalana de la Fibrosi Quística. Our team was supported through funding from the IBEC
program from the Ministerio de Ciencia y Innovación. The authors would like to thank Laura Moya for
helping us perform the experiments, and Eduard Torrents and Laura Moya for critically reading this
manuscript.

REFERENCES
A., M. (1 de Apr de 2005). PubMed. Obtenido de PubMed:
https://www.ncbi.nlm.nih.gov/pubmed/15573386
Adriana Callicó, B. C. (3 de Sept de 2004). scielo. Obtenido de scielo:
http://scielo.sld.cu/pdf/vac/v13n3/vac01304.pdf
Fleming, C. d. (2016). Fisioterapia Respiratoria. Obtenido de Fisioterapia Respiratoria:
http://fisioterapia-respiratoria.com/se-pueden-combatir-la-infecciones-cronicas
IBEC, I. f. (2019). ibecbarcelona.eu. Obtenido de ibecbarcelona.eu:
https://www.ibecbarcelona.eu/es/como-evitar-que-las-bacterias-se-sientan-como-en-
casa/
Masanori Toyofuku, S.-S. Y. (2018). ScienceDirect. Obtenido de ScienceDirect.
O'Sullivan BP, F. S. (30 de May de 2009). PubMed. Obtenido de PubMed:
https://www.ncbi.nlm.nih.gov/pubmed/19403164
O'Sullivan BP, F. S. (30 de May de 2009). PubMed. Obtenido de PubMed:
https://www.ncbi.nlm.nih.gov/pubmed/19403164
PM., Q. (2 de Aug de 2008). PubMed. Obtenido de PubMed:
https://www.ncbi.nlm.nih.gov/pubmed/18675692
Quística, F. E. (2019). fibrosisquistica.org. Obtenido de fibrosisquistica.org:
https://fibrosisquistica.org/que-es-la-fibrosis-quistica/
Sanz DJ, H. J. (1 de Sep de 2017). PubMed. Obtenido de PubMed:
https://www.ncbi.nlm.nih.gov/pubmed/28863137
Torrents, E. (3 de Apr de 2014). NCBI. Obtenido de NCBI:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009431/

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ANNEX

Annex I. Approximate age of onset of clinical manifestations of CF (O'Sullivan BP F. S., 2009)

Annex II. BRANSON Sonicator Machine

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Annex III. Western Blot tank fulfilled with TGS 1x buffer and 2 gels

Annex IV. Homogenizing the sample of sputum with PBS

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Annex V. Pellets obtained after processing the samples

Annex VI. Restreak in a selective media for Pseudomonas of some colonies grown in LB agar
from sputum sample and observable growth

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Annex VII. Pellet resuspended in Sonication buffer during the sonication

Tubs eppendorf [ ] (µg/ml) H2O milli-Q (µl) BSA (µl) Bradford 1x (µl)

1 0 (blanc) 10 0 990

2 1 9,5 0,5 990

3 2 9,0 1 990

4 4 8,0 2 990

5 8 6,0 4 990

PA14 7,0 3 990

Sputum 7,0 3 990

Annex VIII. Concentrations for the quantification with Bradford

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Annex IX. Well plate for the protein quantification with Bradford. From 0 to 8 in the eppendorfs
on the rack and from A to E respectively are the different concentrations of BSA, F is PA14 and
G is the sputum of the patient with CF Each sample with three replicates to ensure the results

Annex X. Proteins running through the gel due to the electrophoresis inside the tank with the
buffer. We can see the path of each sample thanks to the Laemmli buffer that dyes them (first
well is PA14, next to it is the sputum sample, and next is the ladder; 2 wells are empty, and again
the same order)

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Annex XI. Preparation of the Criterion

Blotter Turbo™ Transfer System to transfer
the proteins from the gel to the membrane

Annex XII. Milk blocking both membranes

separated in different containers

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Annex XIII. Protocol for the DNAse treatment to completely eliminate DNA from our sample

Annex XIV. DNA absence test protocol

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Annex XV. cDNA synthesis protocol

Annex XVI. Plate layout and calculations of the MasterMix to prepare the 96-well reaction plate
for the qRT-PCR
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Annex XVII. Protocol to set the PCR

Annex XVIII. Screen and Plot during the process of the qRT-PCR that tells how it is going and
in which part of the cycles is

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Annex XIX. RNA quantification curve of one of the replicates of the sample with sputum from a
patient with CF. 230nm show the contamination, 260nm show the quantity of RNA, and from
290nm ahead it shows the quantity of proteins

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