Lab 4 8/3/2023

Analysis of Gene Expression in Plant

•Aim
Measurement of stress-induced changes by analysis the expression of genes
implicated in plant stress tolerance (PAL, CAT, and PR-1).

•Principle
Plants are constantly exposed to a variety of biotic and abiotic stresses. To survive
such stresses, plants have evolved intricate mechanisms to perceive external
signals and activate optimal responses to environmental conditions. At molecular
level, the perception of extracellular stimuli and subsequent activation of
appropriate responses require a complex interplay of signaling cascades. In defense
against pathogens, salicylic acid “SA” is a key component of the signal
transduction pathway that activated resistance against many plant pathogens,
including fungi, bacteria, and viruses.
The gene encoding for phenylalanine ammonia-lyase “PAL” is believed to be
activated by the jasmonic acid “JA”/ethylene signaling pathway in context of
induced plant defenses. PAL1 is the first enzyme in phenylpropanoid biosynthesis
pathway, which provides precursors for lignin and phenols, as well as for SA.
PR proteins such as β-1,3-glucanase “PR2” and chitinase “PR3”, known to disrupt
the fungal mycelial wall, can be induced by SA as well as by pathogenic attack.
Plant induced PR gene expression and stress tolerance are mediated by a complex
set of signal transcription pathways with H2O2 as a common signal molecule in the
activation stress responses.

•Materials
Item Description
Plastic wares Rack, waste container, eppendorfs, tips, micropipettes, ice box
Glass wares Beaker
Other tools Marker, lab coat, gloves, stop watch, parafilm
Equipment -Cooling centrifuge [Serial no. 179555 – model 3-14KS. Sigma company]

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 -Thermal block [Serial no. 2B0533014 – type TB1. Biometra company]
-Gel electrophoresis system [Serial no. 2403301. BioRAD company]
-UV Spectrophotometer
-Refrigerator [Serial no. 179555. White Whale]
-Vortex mixer [Serial no. B091100654. Grant-Bio PV-1 company]
-BIORAD thermal cycler [serial no. 2609281]

Reagent Uses
DEPC (Diethylpyrocarbonate) -An alkylating reagent that binds to the histidine residues
of the catalytic site of the RNases, destroying their
enzymatic activity, and produces RNase-free solutions.
-DEPC water is responsible for deactivation of RNases to
maintain the integrity of RNA during extraction.
RNAse inhibitors Protects RNA from degradation during isolation and
purification and in all downstream applications such as
reverse transcription into cDNA by RT-PCR.
Ethanol or Isopropanol Storage of RNA.
RNase-free buffer Re-suspension of RNA.
Guanidinium isothiocyanate Powerful protein denaturant used during cell lysis
because of its ability to irreversibly inactivate degrading
nucleases (RNases).
Acidic phenol/chloroform Partitioning of RNA into aqueous phase for separation.
TRIZOL reagent Maintains the integrity of the RNA, while disrupting cells
and dissolving cell components.
Isopropyl alcohol -RNA recovery by precipitation.
-Yields proteins from organic phase.
Ethanol -Yields DNA from the interphase by precipitation.
•75% ethanol for washing.
Ice cold PBS Isotonic buffer solution help in getting rid of cell debris
and medium based contaminants without decreasing RNA
purity.
β-meracptoethanol -Reduce disulfide bonds and act as a biological
antioxidant by scavenging hydroxyl radicals.
-Reducing agent that disrupts tertiary and quaternary
protein structures and unfold native proteins.

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10X MOPS buffer
Sample buffer
10X loading buffer
Electrophoresis buffer
RNA extraction buffer
Formaldehyde
Formamide
Glycerol in loading dye
Bromophenol Blue in loading dye Color tracker or marker to monitor the end of
•Methods “for RNA extraction”
Group A samples:
•I1 “Infected 1”
•I2 “Infected 2”
•S1 “Seedling 1”
•S2 “Seedling 2”
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•200 mM MOPS pH=7.
•50 mM Na-acetate.
•10 mM EDTA pH=8.
-Maintain pH levels, inactivate RNase and enzymatic
activity.
•200 µl 10x MOPS buffer.
•330 µl 37% formaldehyde.
•1000 µl formamide.
•2 µl 0.5 M EDTA pH=8 (final conc 1mM).
•500 µl glycerol (50%).
•475 µl DEPC treated water.
•25 µl (10 mg/ml) ETH. BR (final conc 0.25 mg/ml).
•Few crystals of Bromophenol Blue.
•5x MOPS buffer 300ml.
•37% formaldehyde 44.6ml.
•Distilled water up to 1.5 liter.
•0.2 M borax.
•30 mM EGTA.
•1% (w/v) SDS.
•5 mM dithiothreitol (DTT) was added before use.
-Disrupt secondary structure of RNA.
-Maintain RNA in the denaturation state.
-Protect RNA from RNases.
Act as solubilizing agent and RNA denaturing agent.
Increase sample density to help the sample to sink in the
gel during the run.
electrophoresis run.
-Protocol steps: “We started from the washing step from RNA extraction
protocol-step no.11”
1) Centrifuge at 10,000 rpm for 10 minutes at 4°C.
RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom
of the tube.

2) Remove supernatant then add 1 ml 70% (ice cold) ethanol to RNA pellets. Then
centrifuge (10.000 rpm) 5 min. at 4ºC.

3) Pour off the supernatant.

If the pellet is visible, pipette off any excess ethanol & let air dry for 10-15 min. Pellet should be
almost completely dry, but a little moisture will help it dissolve better.

4) Re-suspend the RNA pellet in 50 μl DEPC treated water (RNase-free water).

30 μl if the pellet size is small, and 50 μl if the pellet size is large. Leave on ice for 10-15 minutes
and then mix by passing the solution up and down through a pipette tip.

5) Measure RNA absorbance at 260 and 280 nm by diluting DNA extract 1:100
with distilled H2O (total vol. 500 μl).

6) Calculate RNA concentration and comment on the purity.

7) Prepare the sample for the gel. [5 μl dye : 5 μl DEPC water : 5 μl sample]

8) Check for integrity and purity by running a sample on native agarose gel.

-Native Agarose Gel Electrophoresis preparation:

•Sample preparation:
Mix the following in an eppendorf:
5 μl sample (containing 5 – 15 μg RNA)
3 μl Sample loading buffer

•Electrophoresis:
1) Place the solidified gel in its place in the electrophoresis tank.
2) Fill in the electrophoresis tank with electrophoresis buffer.
3) Load the prepared RNA samples.
4) Plug in the electrodes and apply 5-10 V/cm of gel (i.e. 50 – 100 volt for 10 cm
long gel).
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5) After the blue front reaches close to gel end, switch off the volt and examine the
gel on a transilluminator.

-On parafilm, add 5 μl dye + 2 μl sample buffer + 5 μl sample then mixing with
pipette. After mixing, load 5 μl from the mixture on each well.

-Calculations:
Purity of RNA absorbance at 260 and 280 nm = 1.8-2

I1 concentration = 3.369 μg/μl

I1 purity = 1.1593

I1 volume to cDNA = 1.5 μl

I2 concentration = 3.443 μg/μl “Approximately = 3.5 μg/μl”

I2 purity = 1.1597

I2 volume to cDNA = 1.5 μl

S1 concentration = 3 μg/μl

S1 purity = 1.185

S1 volume to cDNA = 2 μl

S2 concentration = 3.172 μg/μl

S2 purity = 1.193

S2 volume to cDNA = 1.5 μl

•Expected results
-Appearance of clear and intact RNA that have sharp 28S and 18S rRNA bands on
the gel.

-The 28S rRNA band should be approximately twice as intense as the 18S rRNA
band.
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-This 2:1 ratio (28S:18S) is a good indication that the RNA is intact.

•Real results

•Discussion
-In I2 and I1 samples, they have very low molecular weight smear appearance
which indicates that RNA is completely degraded.

-In S2 sample, there’s DNA contamination and intact RNA is appeared on the gel
“have sharp 28S and 18S rRNA bands with 2:1 ratio and 28S has twice as intense
as the 18S rRNA band”.

-In S1 sample, there’s protein contamination and DNA contamination. Sample has
smeared appearance that lacks the sharp rRNA bands ‘not exhibit 2:1 ratio” which
indicate that RNA is partially degraded.

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 •Trouble shooting
Problem Possible reasons Possible solutions
Low yield of RNA -RNA not solubilized -For solubilization:
completely. •Pippet RNA pellet in SDS or
-Sample not homogenized DEPC-treated water.
completely. •Heat sample at 55º C for 10-15
min.
•Don’t allow RNA pellet to dry
completely.
-For homogenization:
•Make sure to incubate for 5 min
at RT after homogenization.
•Make sure no particulate matter
remains.
Degraded RNA -Improper storage of RNA. -Store isolated RNA at -70º C not
-Sample manipulated too much -20º C.
before the addition of TRIZOL -Process tissue immediately after
reagent. removal from animal.
-Frozen tissue thawed in -Minimize washing steps for cell
absence of TRIZOL reagent. culture samples.
-Add TRIZOL reagent directly to
plates. Don’t trypsinize cells.
-Add frozen tissue to TRIZOL
reagent.
Low A260/280 -Organic solvents “phenol- -Precipitate RNA again with
(<1.65) chloroform” in RNA. ethanol.
-Sample not homogenized with -Make sure not to carry any of
sufficient TRIZOL reagent. organic phase with RNA sample.
-pH of solution is acidic. -Make sure to incubate sample for
-A260 or A280 outside the 5 min at RT after homogenization.
linear range. -Dissolve sample in TE instead of
water.
-Dilute sample to bring
absorbance into linear range.
RNA contains -Part of interphase was removed -Make sure not to take any of
DNA with aqueous phase. interphase with aqueous phase.
-Insufficient TRIZOL reagent -Make sure that original sample
used. doesn’t contain any organic

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 -Cells contained organic
solvents.
-Insoluble materials weren’t
removed before chloroform
extraction.
solvents.
-Remove any particulate material
before chloroform addition.

•Conclusion
-S2 sample is the best to work on as it has clear and intact RNA.

-The protocol was not as successful as expected because of the degraded samples.

•Methods “for Reverse transcription PCR (RT-PCR)”

Samples used:
•S2 with concentration = 3.172 μg/μl
•I2 with concentration = 3.5 μg/μl

-Protocol steps:
1) Add the following into sterile tube “in ice” with the following order: Template
RNA-Primers-water nuclease-free up to 12 μl.

•In tube containing S2 sample:

-Template RNA » 2 μl
-Primers “Oligo&Random” » 1 μl each
-Nuclease-free water » 8 μl

•In tube containing I2 sample:

-Template RNA » 1.5 μl
-Primers “Oligo&Random” » 1 μl each
-Nuclease-free water » 8.5 μl

2) Mix gently then incubate in thermal block at 65º C for 5 min.

3) Spin down and place the tube back on ice.

4) Add the following components on the two tubes in this order: 5X reaction
buffer-RiboLock RNase inhibitor-dNTP mix-RevertAid M MuLV RT.

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 5) Mix gently and centrifuge briefly.

6) For oligo(dT)18 or gene-specific primed cDNA synthesis, incubate for 60 min at

42°C. For random hexamer primed synthesis, incubate for 5 min at 25°C followed
by 60 min at 42°C.
For GC-rich RNA templates the reaction temperature can be increased up to 45°C.

7) Terminate the reaction by heating at 70°C for 5 min.

For S2 tube For I2 tube

-Template RNA » 2 μl
-Primers “Oligo&Random” » 1 μl each
-Nuclease-free water » 8 μl
-5X reaction buffer » 4 μl
-RiboLock RNase inhibitor » 1 μl
-dNTP mix » 2 μl
-RevertAid M-MuLV RT » 1 μl
-Template RNA » 1.5 μl
-Primers “Oligo&Random” » 1 μl each
-Nuclease-free water » 8.5 μl
-5X reaction buffer » 4 μl
-RiboLock RNase inhibitor » 1 μl
-dNTP mix » 2 μl
-RevertAid M-MuLV RT » 1 μl

•Trouble shooting
Problem Possible reasons
Low yield or no RT-PCR -Degraded RNA template.
product -Low template purity.
-Insufficient template quantity.
-Incorrect primer choice.
-GC rich template.
Possible solutions
-Always assess the integrity of
RNA prior to cDNA synthesis.
-Sharp 18S and 28S RNA bands
should be visible after denaturing
agarose gel electrophoresis.
-Follow general
recommendations to avoid RNase
contamination.
-Remove trace contaminants by
re-precipitating the RNA with
ethanol and wash the pellet with
75% ethanol.
-Increase the amount of template
to the recommended level.
Following DNase I treatment,
terminate the reaction by heat
inactivation in the presence of
EDTA, RNA hydrolyzes during

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 heating in the absence of a
chelating agent.
-Use the correct primer for the
RNA template.
-Use the random hexamer primer
instead of the oligo(dT) primer
with bacterial RNA or RNA
without a poly(A) tail.
-Ensure sequence-specific
primers are complementary to 3’-
end of the template RNA.
-For GC rich template, increase
the temperature of the reverse
transcription reaction up to 45°C.
RT-PCR product longer RNA template is contaminated To avoid amplification of
than expected with DNA. genomic DNA, design PCR
primers on exon-intron
boundaries.
RT-PCR product in RNA template is contaminated Perform
negative control with DNA. DNase I digestion prior reverse
transcription.

•Methods “for Quantitative Real-Time PCR”

1) Gently vortex and briefly centrifuge DreamTaq PCR Master Mix (2X) after
thawing.

2) Place a thin-walled PCR tube on ice and add the following components for each
25 μL reaction: “Two tubes: Target gene tube, Housekeeping gene tube”

-DreamTaq PCR Master Mix » 12.5 μl

-Forward primer » 2.5 μl

-Reverse primer » 2.5 μl

-Template DNA » 0.1 μl

-Nuclease-free water » 7.4 μl

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 3) Gently vortex the samples and spin down.

4) When using a thermal cycler that does not contain a heated lid, overlay the
reaction mixture with 25 μL of mineral oil.

5) Perform PCR using the recommended thermal cycling conditions outlined

below:

Step Temperature Time Number of cycles

Initial denaturation 95 1-3 min 1
Denaturation 95 30 s
Annealing Tm-5 30 s 25-40
Extension 72 1 min
Final extension 72 5-15 min 1

Note: For group A » we added RNA instead of cDNA. We then added

cDNA on the tube containing RNA to see the results.

-Primers used for real time RT-PCR assays

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•For dry lab part “getting the product length”:
-Open Primer Blast from NCBI.

-In primer parameters section, enter the forward and reverse primers for the gene
“written in the table above”

-In Primer Pair Specificity Checking Parameters section, choose Refseq mRNA
database.

-Then “Get Primers”

-Another window will appear showing different results for the product lengths.

-Repeat the steps but with choosing Refseq representative genomes database.

-Then repeat the same steps for each gene.

•Conclusion:
Different product lengths may indicate:
-DNA contamination.
-The plant may have viral infection that required Disinfection protocol
to be done first before working on the rest of protocols.

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