1In-Person Lab #3: RNA Interference, RNA Isolation and Introduction to RT-PCR
Important: Experiments run in a one-and-half-week block
Labs running between: March 23 – April 1, 2022
Week #1
BIOC-Lab51 (Day 1): March 23, 2:30 p.m., Biology Building, Room 301
BIOC-Lab52 (Day 1): March 23, 6:00 p.m., Biology Building, Room 301
BIOC-Lab53 (Day 1): March 25, 2:30 p.m., Biology Building, Room 301
BIOC-Lab54 (Day 1): March 25, 6:00 p.m., Biology Building, Room 301
Week #2
BIOC-Lab51(Day 2): March 29, 2:30 p.m., Biology Building, Room 301
BIOC-Lab51(Day 3): March 30, 2:30 p.m., Biology Building, Room 301
BIOC-Lab52 (Day 2): March 29, 6:00 p.m., Biology Building, Room 301
BIOC-Lab52 (Day 3): March 30, 6:00 p.m., Biology Building, Room 301
BIOC-Lab53 (Day 2): March 31, 2:30 p.m., Biology Building, Room 301
BIOC-Lab53 (Day 3): April 1, 2:30 p.m., Biology Building, Room 301
BIOC-Lab54 (Day 2): March 31, 6:00 p.m., Biology Building, Room 301
BIOC-Lab54 (Day 3): April 1, 6:00 p.m., Biology Building, Room 301
Educational Objectives:
By the completion of this lab you should be able to:
i.
Explain the molecular mechanisms associated with RNA interference as well
as the practical uses of siRNA in a research laboratory
ii.
Perform RNA isolation in an efficient and sterile manner such that RNA
degradation does not occur
iii.
Perform the reverse transcription and PCR techniques in an organized and
efficient manner; Explain the individual function of each reaction component
iv.
Operate the NanoDrop, Thermocycler, Electrophoresis equipment and
SynGene Imaging System in a safe and efficient manner
v.
Use results from your RT-PCR reaction to semi-quantify the expression levels
of PTEN and GAPDH from HEK293 cells that were treated with siPTEN,
siGAPDH, negative control siRNA or untreated cells
2
Background:
“Total RNA” is a common term used to describe the entire complement of RNA
molecules found in any given cell and includes the three major types of RNA molecules;
messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). As a quick
review, mRNA is transcribed as a single-stranded molecule containing complementary
ribonucleotides from gene-containing template DNA. Transfer RNA is involved in protein
synthesis within the ribosome (composed of rRNA), and functions by converting the
information carried by mRNA into a corresponding amino acid sequence. In addition, each
cell contains small RNA molecules that play a variety of roles in regulatory function. Some
examples include small nuclear RNA (snRNA) and micro RNA (miRNA), which are
involved in the maturation of mRNA and the regulation of gene expression, respectively.
Messenger RNA is the key link between the information stored within a gene and
the expression of that information via protein synthesis. This relationship is typically
referred to as the central dogma of molecular biology, in that DNA is transcribed into
mRNA and then translated into protein. This flow of genetic information occurs in all cells
where the genetic carrier is double-stranded DNA. mRNAs serve a central function in the
transport, regulation and translation of information from DNA nucleotides to a sequence
of amino acids that ultimately form fully functional proteins. Throughout this elegant
process, mRNA expression is controlled by a number of regulatory proteins in an effort to
manage multiple events that affect the cell cycle such as level and/or stability of mRNA in
specific cell types, ability to form functional proteins following post-transcriptional
modification and even mRNA translocation within the cell. Overall, cellular control of
gene expression is a complex process that involves significant interaction between
regulatory proteins and/or elements and multiple RNA molecules.
In this experiment, we will be investigating a technique known as RNA interference
(RNAi). The in vivo process of RNAi was initially discovered in plants, where it was
originally described as post-transcriptional gene silencing. Also known as RNA silencing,
this phenomenon has since been described in a wide variety of eukaryotic organisms and
has evolved into a powerful tool used to artificially down-regulate the expression of
specific target genes in organisms as diverse as Caenorhabditis elegans, Drosophila
melanogaster and Mus musculus.
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RNAi is a natural process that is triggered by double-stranded RNA precursors that
are present during viral infection of mammalian systems. These precursors vary in length
and are processed into short RNA duplexes of 21 to 28 nucleotides in length. The 21-28
nucleotide duplexes in turn are responsible for gene silencing by specific mechanisms that
vary in different systems. Some examples include mediation of translational repression,
guidance of mRNA degradation, and alteration of chromatin structure. For an in depth
review of gene silencing using RNAi, you can refer to the following publication: Meister
et al. Nature 431: 343-349 (2004).
One particular RNAi mechanism involves the use of small interfering RNA
(siRNA) molecules. siRNAs consist of 19-21 base pair duplexes, containing both the sense
and anti-sense strands with two nucleotides that overhang at the 3’ end. When used as an
experimental technique, siRNAs are translocated into mammalian cells using a transfection
reagent containing a combination of polyamines that allow for the transfection of small
RNA molecules into the cytoplasm without cytotoxic impacts to the cells. Upon entering
the cell, siRNA molecules bind to a nuclease complex to form an RNA-inducing silencing
complex, also known as RISC. Following activation of the ATP-dependent RISC complex,
the siRNA molecule unwinds. The conjoined siRNA and nuclease complex targets the
homologous transcript (i.e. complementary RNA strand) by base pair matching. This
affinity to complementary mRNA allows for the gene-specific splicing of targeted mRNA
molecules by the RISC complex. The mRNA is cleaved approximately 12 – 15 base pairs
from the 3’ end of the siRNA molecule, thereby causing a reduction in cellular gene
expression levels. This silencing reaction is very powerful, and it has been suggested that
cellular replication of the siRNA molecule may be occurring.
Figure 1: RNAi triggered mRNA molecules by the RISC complex. Image adapted from the Silencer®
siRNA starter Kit from ThermoFisher.
4
Your first task is to isolate total RNA from human embryonic kidney (HEK293)
cells that have been treated with either siPTEN, siGAPDH (Glyceraldehyde 3-phosphate
dehydrogenase), negative control siRNA for 48 hours or an untreated cell control. During
the RNA isolation process, there are a number of factors that you must consider in order to
obtain the expected results. The chemical structure of RNA involves an additional hydroxyl
(OH) group in the ribose sugar, which makes the molecule sensitive to hydrolysis and
therefore degradation. In addition, since RNA is single-stranded, it is less ‘protected’ than
its’ DNA counterpart. Furthermore, RNase enzymes are abundant in the laboratory
environment, and even a small amount of RNase contamination can wreak havoc on your
experiment. To reduce the possibility of contamination, all solutions, glassware and plasticware will be pre-treated to denature and/or destroy ribonucleases. Solutions used in the
isolation of RNA are pre-treated with diethylpyrocarbonate (DEPC), which inactivates
ribonucleases. However, not all buffers can be treated with DEPC (i.e. Tris based
solutions), therefore extreme care must be taken with these buffers. Glassware will be
baked at 400 ˚C for at least four hours because the autoclave is unable to destroy RNases.
Tips and tubes used in this lab have been certified as “RNase and DNase Free” so extreme
care must be taken to ensure that you do not contaminate tips and tubes. Finally, the major
source of contaminating RNase comes from your hands. Therefore, it is absolutely
mandatory that gloves be worn at all times during the isolation and preparation of RNA.
During the isolation of RNA from 293 cells you will be using an RNeasy Mini kit
that is commercially available from QIAGEN. Following RNA isolation, you will generate
cDNA, and perform PCR to determine the expression levels of GAPDH and PTEN from
the siRNA treated cells and the experimental control. It is your responsibility to understand
how each of these techniques is performed and explain the function of individual reaction
and procedure components.
Safety Requirements and Warnings:
•
Good laboratory practices require that you wear a lab coat, disposable gloves and
safety glasses at all times.
5
•
It is important for you to read the labels on the waste containers!
The QIAGEN RNAeasy kit we are using for RNA extraction contains the following
warning label:
•
Make sure you dispose of liquid waste correctly! If you are not sure, ask your GA
where to dispose of the liquid waste.
•
As good laboratory practice is a must during this experiment, make sure you follow
all procedures and pay attention while inside of the laboratory.
•
Before you leave, make sure you dispose your waste into an appropriately labelled
containers and decontaminate all pipettes individually and your lab bench with 70%
ethanol. Ensure you leave your working area the way you found it – everything should
be neatly organized and the content of the small biohazard containers should be moved
into the large biohazard box, the tip boxes (keep possible contamination in mind and
try to prevent it) are tightly closed, all instruments present at your work-station are turn
off, gel boxes are cleaned, gently wiped dry and returned back to where you found
them, etc. To summarize, if your work area does not look the same after you leave as
it did when you came in, you and your lab-partner participation marks will be deducted.
6
Procedure Overview:
Day 1: RNA isolation, RNA quantification and cDNA synthesis
Day 2: Semi-quantitative RT-PCR
Day 3: Agarose gel electrophoresis
1 Week (7 days) after Day 3 – Lab report due on Blackboard
Procedures:
Day 1
Important information:
As you are working with RNA! All spins must be performed at room temperature
and maximum speed. All steps must be performed quickly to prevent RNA degradation.
Work at the same pace as one other group of two so that you can centrifuge with them and
not have to create weight tubes for balancing the centrifuge.
Part A: RNA Isolation
1. All reagents you need to perform the RNA isolation from mammalian HEK293
cells will be placed on your bench with the exception of the tissue culture plates
containing HEK293 cells. These cells have been transfected with one of three types
of siRNA: siPTEN, siGAPDH or negative control siRNA. An additional, untreated
control plate will be included as well.
2. Your GA will give you one 35mm plate of cells that has been treated with siPTEN,
siGAPDH, the negative control or untreated control. You group will be only
working with one of the four experimental samples – make sure you note which
one!
3. Aspirate the media from the plate using the vacuum station and wash the cells by
carefully adding 1 mL of room temperature 1X PBS to the side of your plate. Be
careful, as HEK293 cells lift-off very easily, thus don’t add the PBS to the middle
of the plate, as you will dislodge and lose cells. Gently tilt the plate front to back
once for a complete wash.
7
4. Aspirate all of the 1X PBS from the plate.
5. Return to your bench.
6. Add 350 µl of lysis buffer (RLT contains beta mercaptoethanol) to your plate.
Gently tilt the plate front to back to ensure that all cells are lysed.
7. Use a cell scraper to gently collect the cellular lysate in a pool. Aspire the cell lysate
using a P1000 set to 500 µl. Make sure no cell clumps are visible prior to moving
to step 8. If cell clumps are present, break them apart by pipetting up-and-down
slowly.
8. Transfer disrupted cells to a purple QIA shredder column within a 2 ml collection
tube. Label the side of the tube with your initials and plate identity (PTEN,
GAPDH, NEG or cells).
9. Centrifuge at max speed for 2 minutes – keep the flow thru
10. Add 1 volume (500 µl) of 70% ethanol to the homogenized lysate in the 2 ml tube
and pipette gently to mix.
11. Transfer up to 700 µl of the sample (from step 10) into a pink RNeasy spin column
placed in a 2 ml collection tube.
12. Centrifuge for 15 seconds at max. Discard the flow-thru into the appropriate waste
container beside the centrifuge.
13. Centrifuge successive aliquots in the same column. Discard the flow thru into the
appropriate waste container after each spin.
14. Add 700 µl Buffer RW1 to the spin column. Close the lid and spin for 15 seconds.
Discard the flow-thru into the appropriate waste container next to the centrifuge.
15. Add 500 µl Buffer RPE to the spin column. Close the lid and spin for 15 seconds.
Discard the flow-thru into the appropriate waste container next to the centrifuge.
16. Add another 500 µl Buffer RPE to the spin column. Close the lid and spin for 2
minutes. Carefully transfer the spin column to a labeled 1.5 mL tube without
allowing any flow-thru liquid to touch the column. Discard the flow-thru into the
appropriate waste container next to the centrifuge.
17. Add 30 µl of DEPC-treated water to the center of the spin column membrane. Close
the lid and spin for 1 minute to elute the RNA.
8
18. Put your tube of RNA on ice immediately. Keep your RNA on ice until all
procedures are completed (i.e. keep RNA in ice bucket when transporting to
NanoDrop and back to the lab). Remember that RNA degrades easily. Be careful
not to contaminate your purified RNA!
Part B: RNA Quantification
1. Go to the NanoDrop station and open the NanoDrop software on the computer
desktop. Click on the Nucleic Acid application module.
2. To initialize the spectrometer load 2 µl of DEPC-treated water onto the lower
measurement pedestal. Carefully close the sampling arm and click “OK”. After
clicking “OK”, the message “Initializing Spectrometer – please wait” will appear.
When this message disappears the instrument will be ready for use. Wipe the water
sample off the pedestal and arm using a KimWipe.
3. On the upper right of the screen change the nucleic acid type from DNA to RNA.
4. Before making a sample measurement, a blank must be measured and stored. Load
2 µl of DEPC-treated water onto the pedestal and close the sampling arm. After
clicking “Blank”, the measurement will be taken and a straight line will appear on
the graph. Wipe the blank from the pedestal and arm using a KimWipe.
5. Analyze an aliquot of DEPC water as though it were a sample. Load 2 µl of water
onto the pedestal, close the sampling arm, and take the measurement by clicking on
the “Measure” button. The result should be a spectrum with a relatively flat
baseline. Wipe the water aliquot off the pedestal and arm using a KimWipe.
6. Analyze the concentration of your purified RNA. Load 2 µl of RNA sample onto
the pedestal, close the sampling arm, and take the measurement by clicking on the
“Measure” button. Wipe your sample off the pedestal and arm using a KimWipe.
7. Record the concentration of your sample (ng/µl), the absorbance at 260 and 280
nm, and the 260/280 ratio. Remember to comment on the concentration and purity
(260/280) of your sample in your lab report.
8. Return back to the lab and based on your sample concentration determine the
volume of RNA required for reverse transcription (Part C).
9
Part C: Reverse Transcriptase (First Strand Synthesis)
1. Acquire the box for your group from the -20˚C freezer and place on ice
immediately. After thawing, spin the reagents for 10 seconds and put back on ice.
2. Anneal primer to template RNA. Combine the following components in the 0.2 ml
PCR tube that your Oligo dT primer is already in. Remember to label your tube
well.
1 µl
Oligo dT primer
1 µl
dNTP mix (10 mM)
2.875 µl
Total RNA (2 µg) – use a maximum of 11 µl
8.125 µl
DEPC-treated water
13 µl
Total volume of reaction
3. Close the PCR tube lid and gently tap the bottom of the tube to mix the reagents.
Spin the PCR tube for 10 seconds. Incubate at 65˚C for 5 minutes and then incubate
on ice for at least 1 minute.
4. Prepare RT reaction mix. In a different PCR tube combine the following reagents
in the order listed below:
4 µl
5X SSIV Buffer (tube labelled as 5xSSIV)
1 µl
DTT (100 mM)
1 µl
Ribonuclease Inhibitor
1µl
Superscript IV Reverse Transcriptase (tube labelled as RT)
5. Cap the tube you assembled in step 4, mix and briefly centrifuge the contents.
6. Add RT reaction mix to the annealed RNA. Incubate the combined reaction mixture
at 50˚C for 10 minutes. Inactivate the reaction by incubating it at 80˚C for 10
minutes. Store your first-strand cDNA sample at -20˚C until your next lab session.
End of Day 1
10
Day 2
Part D: RT-PCR
Note: In order to complete this lab within the allotted 3 hours your RT-PCR reactions
MUST be assembled in 30 minutes or less.
1. Retrieve your cDNA sample from Day 1 and reagents for Day 2 from -20˚C freezer
and place on ice immediately. Use reagents box as a rack to prepare your samples
in.
2. Pre-label 14 thin-walled PCR tubes as described below:
PTEN
GAPDH
P18
G18
P20
G20
P22
G22
P24
G24
P26
G26
P28
G28
P (-)
G (-)
3. Prepare two separate master mixes for all of the PCR reactions, one with GAPDH
primers and one with PTEN primers. Follow the recipe below for each master mix:
56 µl
ddH2O
70 µl
2X PCR Master Mix (contains dNTPs, Taq DNA Polymerase,
MgCl2, reaction buffer)
7 µl
Primer Mix (labeled GAP or PTEN and contain both the forward
and reverse primers – only use one per master mix)
133 µl
Total volumes required for seven 19 µl reactions
4. Tap the bottom of the tube to mix and then briefly spin down the contents.
11
5. Add 19µl of the appropriate master mix to each of your labeled PCR tubes.
6. Add 1µl of your cDNA template to 12 of the 14 tubes. Do not add template to the
negative control tubes that you labeled as P (-) and G (-). Instead, add 1 µl of water
to the negative control tubes.
7. Close all of the tubes and tap the bottom of each tube to mix the contents. Briefly
spin down the tubes. Keep all tubes on ice.
8. Once all groups have assembled their reactions, bring your tubes to your GA to be
placed in the thermocycler.
9. At the end of the extension time during the 18th PCR cycle, your GA will pause the
PCR machines and remove all the tubes labeled G18 and P18 (these are run in
separate PCR machine as the programs are different for each primer set). The lid
will then be closed and your GA will resume the PCR cycles. This process will be
repeated for each cycle that you will be examining (i.e. cycles 18, 20, 22, 24, 26
and 28 for GAPDH and PTEN).
10. Collect each sample into a plastic bag labeled with your group names and section
number. Put the baggie in the bin that is labeled for your section in the freezer for
your next day in the lab.
11. Your negative control reactions for P (-) and G (-) will be removed with your 28th
PCR cycle.
PCR program for PTEN: 94-2m,[ 94-30s, 62-30s, 72-1m ]28 cycles, 72-7m, 4∞
PCR program for GAPDH: 94-2m,[94-30s, 56-30s, 72-1m]28 cycles, 72-7m, 4∞
End of Day 2
12
Day 3
Part E: Agarose gel electrophoresis
Note: Two groups of two students will run their samples on one double (2X20) well large
format gel. You must load your gel relatively quickly.
1. Carefully transfer a pre-made 1% TAE agarose gel into the tray of the apparatus.
2. Place your gel tray in the electrophoresis unit and submerge under 3 – 5 mm of 1X
TAE buffer.
3. Retrieve your PCR reactions from last week and organize them in the 96 well rack
on your bench. Thaw your samples and briefly spin down each reaction.
4. Add 4µl of 6X Gel Loading buffer to each reaction tube and gently tap the bottom
to mix, and then briefly spin your tubes.
5. Load entire sample of the 1kb DNA Ladder (Frogga Bio) provided and set up your
tubes to match the gel loading guide below
Lane
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Sample
Empty
G18
G20
G22
G24
G26
G28
G (-)
Empty
6 µl DNA ladder
Empty
P18
P20
P22
P24
P26
P28
P (-)
Empty
Empty
6. Carefully load all of your samples into the specified lanes. If you load the wrong
sample into the wrong lane make a note of it.
13
7. Once your gel is loaded put the lid on the apparatus (make sure you are running
from black to red) and plug the leads into the power supply.
8. Turn on the power supply and select your voltage to be constant at 100V.
9. Continually check on the progress of the electrophoresis until the purple dye front
migrates half ways to the edge of the 2nd set of wells and/or the middle of the bottom
part of the gel tray (this takes approximately 1.5 h). Turn off the electric current
and remove the leads and lid from the gel tank.
10. Use the SynGene Imaging system to capture a digital picture of your gel. Your GA
will upload the images onto Blackboard under your lab group folder.
11. Discard your gel in the container in the fume hood, wash your tray with water gently
dry the gel box prior to placing it back where you found it.
End of Day 3
siRNA RT-PCR Representative Data Set
• Use the information and results provided here and compare to your results
• You should comment on the following (this is only a basic guideline):
– Did you get good results from the RT-PCR?
– Did you get the expected bands, why or why not? (there are MANY possibilities)
– Did you get any non-specific bands, what could they be?
– Comment on anything else that is important to your experiment
– Do the values from your quantification (using ImageJ, see below) make sense?
– Do you see a gradual increase in band intensity with each PCR cycle?
– How do your results compare to the posted data set?
– Explain the difference between semi-quantification (what you did) and full
quantification of gene expression (i.e. ways to optimize this experiment)
– Did siRNA treatment work?
– If no, what could have caused it to fail?
– If yes, what could be done to optimize the experiment even more?
• Remember to include any additional information that you think is relevant to the
discussion of your results
• **NOTE: The DNA ladder shown here is the 1kb Fermentas ladder NOT the Froggabio ladder
you used, so you will need to label your bands according to the ladder we used in the lab, not the
one provided in this representative data set and include it in your figures **
The amplified DNA bands will be quantified using ImageJ software, which can be accessed
through http://rsb.info.nih.gov/ij/ You can either select “Run ImageJ in Browser!” option from the
list for your analysis or download this software to your computer. You will need your images as
jpg and opened to load it up for analysis. This software allows you to relatively (no absolute values)
quantify DNA bands from your semi-quantitative RT-PCR. The quantification reflects the relative
amount as a ratio of each DNA band relative to untreated samples (labelled as Raw volume in the
representative date tables).
To help you out and get you started below are some ImageJ very basic how-to:
Set measurement criteria in ImageJ
Under “Analyze” menu select “Set Measurements”. From the checkboxes have ONLY the “grey
mean value” checked.
Open the image – copy paste it into the ImageJ program
siRNA RT-PCR Representative Data Set
Zooming in and out in your image in ImageJ – shift + shift –
Define a selection as a region of interest
Use rectangle
Measurements – make sure same selection box is used for all bands analyzed. Make sure it fit the
biggest band. If you close it or change it, you will have to start over again. Place the frame on the
first band, centre the band inside the frame of the box and use the ctrl M or alternatively click
“measure” under the “analyze” menu. This will open the measurement window and display your
data in order. Move the frame to the next lane and make measurement for that band and across all
bands in your sample. Keep in mind that you will also need to take a background measurement
with the same frame. Record this value. Will need to use it in your calculation.
While you are working through the data analysis, you can move the values into Excel – use for
calculations, creating graphs and tables.
More info can be obtained from https://imagej.nih.gov/ij/docs/menus/analyze.html
2
siRNA RT-PCR Representative Data Set
3
siRNA RT-PCR Representative Data Set
4
Biotechnology siRNA RT-PCR Lab Marking Scheme ( /20)
Introduction – 3 marks
Materials and Methods
As per lab manual (List any protocol changes in this section)
Results – 12 marks (total)
**include legends and labels/titles for all tables, graphs and gel images**
Written component – 2 marks
– Do not repeat lab manual methods/protocols
– Should have a brief paragraph as intro into each result section
RNA Isolation – 1 mark
– Type of sample you received (siNEG, siPTEN, siGAPDH or untransfected cells)
– Table containing RNA concentration (ng/μl) and purity results (260, 280, 260/280)
First strand synthesis, RT-PCR and gel images – properly labelled (4 images total) – 2 marks
– Sample calculation for RNA required for reverse transcription (μl)
– each gel image properly labelled – includes lane assignment, # of PCR cycles, band sizes on the
1kb ladder, etc. and/or posted representative data set (for each siNEG, siPTEN, siGAPDH and
untransfected cells with all cycles and both primer sets)
– What should be labeled for the gel: DNA ladder (mark some of the bands, not all of them if it
doesn’t fit), GAPDH and PTEN Primers (which side is which), arrow pointing to size of PTEN
and GAPDH bands
Graphs (4 in total) of amplification – based on the densitometry analysis (ImageJ values for
amplified DNA bands (y-axis) vs. PCR cycles (x-axis) – 1 mark
o Compare your data (from the class, mention which one is actually your group’s result) to
posted data set (0.25 marks each)
o Analyze all experimental data obtained during your lab section (0.25 marks each) and include
graphs for all of them
o Include both sets of primers on the same graph (GAPDH primers and PTEN primers)
Table of excel densitometry values you generated using ImageJ software for all PCR cycles and
both primer sets and all 4 samples (siNEG, siGAPDH, siPTEN and untransfected) or if only
partial results were obtained, analyze what is available or analyze representative data – 0.25
mark each
Sample calculations Required – 2 marks
ONE sample calculation for determining the normalization factor for ONE PCR CYCLE (this
can be for NFsiPTEN or NFsiGAPDH)
– NFsiPTEN= si(-)_GAPDH primers/si(-)_PTEN primers
– NFsiGAPDH = si(-)_PTEN primers/si(-)_GAPDH primers
1
ONE sample calculations for relative measure of expression (GAPDH or PTEN) due to siRNA
for ONE PCR cycle for YOUR experimental results
– Equations for determining the relative expression for:
siGAPDH = siGAPDH_GAPDH primers/siGAPDH_PTEN primers x NFsiGAPDH
si PTEN = siPTEN_PTEN primers/siPTEN_GAPDH primers x NFsiPTEN
Table summarizing the expression levels for ALL PCR cycles for siGAPDH and siPTEN from
for all the cycles for YOUR experimental sample (NOTE: if you were given siNEG, you should
also include the relative measure of expression for both siGAPDH and siPTEN using your
experimental NFs from your siNEG samples) – 1 mark
Discussion – 5 marks
– Conclusion and final remarks is part of this section
– Comment on RNA isolation and purity (compare to ‘good’ values)
– Comment on graph trends (i.e. should see gradual increase in amplification with increasing
PCR cycles for the negative control and lower with siRNA treatment)
– State results from experimental gel image (If your experiment did not work provide
scientific explanation as to why – ‘human error’ is not a feasible explanation)
– Compare results to posted data sets and describe the effect of siRNA treatment (need to
state actual calculated values here)
– Brief explanation why a normalization factor had to be used and that we used GAPDH for
this (also comment on why the determination for GAPDH levels are not accurately reliable)
– How these results could be better
– Controls, sources of error, replicate experiments etc.
Reference
-2 if missing and/or not included in text
2
siRNA RT-PCR Representative Data Set
• Use the information and results provided here and compare to your results
• You should comment on the following (this is only a basic guideline):
– Did you get good results from the RT-PCR?
– Did you get the expected bands, why or why not? (there are MANY possibilities)
– Did you get any non-specific bands, what could they be?
– Comment on anything else that is important to your experiment
– Do the values from your quantification (using ImageJ, see below) make sense?
– Do you see a gradual increase in band intensity with each PCR cycle?
– How do your results compare to the posted data set?
– Explain the difference between semi-quantification (what you did) and full
quantification of gene expression (i.e. ways to optimize this experiment)
– Did siRNA treatment work?
– If no, what could have caused it to fail?
– If yes, what could be done to optimize the experiment even more?
• Remember to include any additional information that you think is relevant to the
discussion of your results
• **NOTE: The DNA ladder shown here is the 1kb Fermentas ladder NOT the Froggabio ladder
you used, so you will need to label your bands according to the ladder we used in the lab, not the
one provided in this representative data set and include it in your figures **
The amplified DNA bands will be quantified using ImageJ software, which can be accessed
through http://rsb.info.nih.gov/ij/ You can either select “Run ImageJ in Browser!” option from the
list for your analysis or download this software to your computer. You will need your images as
jpg and opened to load it up for analysis. This software allows you to relatively (no absolute values)
quantify DNA bands from your semi-quantitative RT-PCR. The quantification reflects the relative
amount as a ratio of each DNA band relative to untreated samples (labelled as Raw volume in the
representative date tables).
To help you out and get you started below are some ImageJ very basic how-to:
Set measurement criteria in ImageJ
Under “Analyze” menu select “Set Measurements”. From the checkboxes have ONLY the “grey
mean value” checked.
Open the image – copy paste it into the ImageJ program
siRNA RT-PCR Representative Data Set
Zooming in and out in your image in ImageJ – shift + shift –
Define a selection as a region of interest
Use rectangle
Measurements – make sure same selection box is used for all bands analyzed. Make sure it fit the
biggest band. If you close it or change it, you will have to start over again. Place the frame on the
first band, centre the band inside the frame of the box and use the ctrl M or alternatively click
“measure” under the “analyze” menu. This will open the measurement window and display your
data in order. Move the frame to the next lane and make measurement for that band and across all
bands in your sample. Keep in mind that you will also need to take a background measurement
with the same frame. Record this value. Will need to use it in your calculation.
While you are working through the data analysis, you can move the values into Excel – use for
calculations, creating graphs and tables.
More info can be obtained from https://imagej.nih.gov/ij/docs/menus/analyze.html
2
siRNA RT-PCR Representative Data Set
3
siRNA RT-PCR Representative Data Set
4
IMPORTANT INFORMATION
We used an untreated si GAPDH
Concentration of our sample (ng/μ), the
absorbance at 260 and 280nm, and the
260/280 ratio values are in the figure
below
1/18/2005 3:27 PM
Default
350
Sample
Type
λ
Sample
ID
RNA-40
Sample # 7
Abs.
A-260 10 mm path
A-280 10 mm path
260
Exit
17.389
17.389
8.505
260/280 2.04
260/230 2.17
ng/uL 695.6
Remember to comment on the concentration
and purity (260/280) of
the sample.
for materials and methods, please
Cr
write
As per lab manual”. Do not
сору
the method’s written in the lab manual
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