Molecular Biology Labs 12
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Background:
The reverse transcriptase polymerase chain reaction (RT-PCR) is used to make
complementary DNA (cDNA) copies of RNA molecules. This procedure has a number of
important applications, including cloning mRNAs, amplifying ends of partial cDNAs,
generation of cDNA libraries, and measuring gene expression when only small amounts
of RNA are present.
There are 2 basic steps to any RT-PCR reaction; reverse transcription (RT) and PCR.
Reverse transcription is simply RNA dependent DNA synthesis that occurs in several
steps. First, an oligonucleotide primer binds to the RNA strand. The reverse transcriptase
then binds to the primer and synthesizes a first strand of cDNA that is complementary to
the RNA. Third, the original RNA strand is degraded by RNAse H activity and the
second strand of cDNA is synthesized by nick translation to produce double stranded
cDNA. The second step in RT-PCR is simply a PCR reaction to amplify the cDNA into
many copies.
The enzyme reverse transcriptase was discovered in 1970 by Howard Temin and David
Baltimore who won the Nobel Prize for their work. There are 2 important examples of
reverse transcription in nature. The first is retroviruses, small viruses such as HIV that
contain an RNA molecule as their genome. When a retrovirus infects a cell, the RNA and
attached reverse transcriptase molecules enter the cytoplasm and quickly synthesize
cDNA molecules complementary to the virus RNA. The cDNAs enter the host cell
nucleus and integrate into the cellular DNA. The integrated retrovirus genome, called the
provirus, continually produces virus RNA. This RNA is packaged into protein capsids
and infectious retroviruses are slowly released from the infected cell. Reverse
transcriptases are essential for the retrovirus life cycle.
A second example of reverse transcriptase in nature is the retrotransposon. These are
regions within DNA of eukaryotes that move (transpose) around the genome. A
retrotransposon can contain a reverse transcriptase gene as well as several additional
genes. When the retrotransposon is expressed, the RNA that is produced can be converted
to cDNA by the encoded reverse transcriptase. This cDNA can integrate at other regions
within the host genome. It is amazing that almost 10% of DNA in eukaryotic cells may
be composed of retrotransposons. It is unclear whether this apparently parasitic DNA has
important functions within the cell.
RT-PCR is used frequently in molecular biology for various purposes. There are
essentially 3 methods that are used to prime the synthesis of first strand cDNA.
1. Gene specific primers are used if one knows which RNA needs to be copied. In
this case, only the gene of interest is amplified from the RNA template.
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2. Random hexamers are used if one needs to amplify a large population of RNA
molecules and no sequence information is known. Random hexamers are just
short (6bp) oligonucleotides that bind throughout the diversity of RNA molecules
and prime synthesis of many different cDNA copies. The cDNAs represent all
types of RNA including ribosomal, transfer, small nuclear, and messenger RNAs.
3. Oligo (dT) primers are oligonucleotides consisting of polyT tracts. These tracts
bind to regions of poly A that exist on the 3’ end of most mRNAs and prime
synthesis of cDNAs that are specific for mRNA. No ribosomal or other types of
RNA are amplified. The large variety of different cDNAs reflects the large
number of mRNAs within the cell. If a mixture of this cDNA is subsequently
cloned into plasmid vectors the product is called a cDNA library (because it is
essentially a library containing all of the mRNAs within the cell). Oligo (dT)
primers are also used to perform RT-PCR in order to amplify specific genes from
the total population. This is the objective of the current lab.
There are different types of reverse transcriptase molecules. Mesophilic enzymes have
associated RNAse H activity and they degrade the RNA strand at the same time that they
synthesize the cDNA copy. This is often a disadvantage because the RNAse H can
degrade the RNA strand before it has been completely copied into cDNA. This results in
stubby cDNAs (less than 2 kb) that are not particularly valuable for molecular biology
experiments. A second type of reverse transcriptase has been genetically engineered to
lack RNAse H activity (superscript II is an example that is used in the current lab
experiment). These enzymes make longer cDNA molecules (up to 10 kb) because the
template strand is not degraded. Finally, thermostabile Tth DNA polymerase can be used
to synthesize both first and second strands in the same reaction. While this is convenient,
it is not used frequently because the cDNA molecules are fairly short (2 kb).
RT-PCR reactions are usually performed using several important controls.
1. No reverse transcriptase control: because RNA preparations are often
contaminated with small amounts of cell DNA, there is a chance that the
amplification products are derived from DNA rather than RNA. To check this
possibility, it is customary to perform one reverse transcriptase reaction without
any enzyme. If you can amplify a product from the RNA preparation it likely
contains a significant amount of contaminating cell DNA. The DNA
contamination can be eliminated by digestion with RNAse-free DNAse.
2. Substituting cell DNA for cDNA control: this control uses dsDNA (high
molecular weight cell DNA) instead of cDNA in the PCR reaction. If there is a
problem in the RT-PCR, it helps to determine whether the fault is a poor reverse
transcription (should still see a band) or poor PCR (should not see a band).
3. The no cDNA control: The PCR reaction is ultra sensitive and may amplify traces
of DNA contaminants present in the lab. This control checks for specificity. If any
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product is made without any template cDNA or DNA, it is clear that your
reagents are contaminated.
4. Another important control is to check the quality of your cDNA preparation. This
can be performed by loading 5 to 10 l on a mini gel and separating the
fragments. If the cDNA preparation is good, you should see a smear of 10 to 1 kb
in size. You should see no smear in your no reverse transcriptase control.
Objectives:
The first objective of this lab is to perform a reverse transcription reaction to generate
first strand cDNA from RNA previously purified in lab. The second objective is to use
the first strand cDNA to perform PCR.
Materials:
Samples of total cellular RNA
Reverse transcriptase kit (superscript first strand synthesis system for RT-PCR from
InVitrogen which contains oligo (dT), 10X reaction buffer, MgCl2, DTT, dNTP mix,
superscript II reverse transcriptase, RNAse inhibitor, RNAse H, DEPC-treated water,
control RNA, control primers)
37C water bath
42C water bath
65C water bath
Disposable gloves
Microcentrifuge
Thermocycler
Thin wall PCR tubes
PCR primers
PCR supermix or PCR reaction components
3M sodium acetate
100% ethanol
Micropipetters
Sterile eppendorf tubes, yellow tips, and blue tips, eppendorf racks
Sterile deionized water
Microwave oven
Mini gel apparatus, combs, and power packs
Gel loading buffer
1X TAE running buffer with ethidium bromide
0.8% agarose with ethidium bromide
100 bp molecular weight markers
Camera for gel photography
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Methods:
Measuring the RNA concentration in the sample:
1. Remove your RNA sample from the -70C freezer. Spin tube for 15 min at top
speed in the table top microcentrifuge to pellet the precipitated RNA.
2. After centrifugation, you should see a small white or amber colored pellet on the
bottom side of the centrifuge tube. Carefully pour the supernatant from the tube
and invert the tube on a paper towel to drain. Be careful that the RNA pellet does
not start to slide down the side of the tube. Try to remove most of the ethanol
from the tube.
3. Add 100-400 l of sterile deionized water to the tube and resuspend the RNA by
pipetting through a yellow or blue tip. Observe the solution carefully to confirm
that all of the RNA has been dissolved and that there are no solid flakes of RNA.
This might require several min.
4. Turn on the BioRad Smartspec and set the analysis mode to DNA/RNA. Keep all
of your RNA samples on ice during this procedure. Push the button that says
RNA.
5. Set the blank for the spectrophotometer. Add 800 l of deionized water to a
quartz cuvette and place in the spec. Push the blank button.
6. Remove 10 l of RNA suspension and add to an eppendorf tube containing 1000
l of deionized water. Mix well by shaking the tube.
7. Add the contents to the cuvette and measure the absorbance and record the value.
Remove the cuvette and decant the sample. Briefly rinse the cuvette with water
before analyzing the next sample.
8. When finished, wash the quartz cuvette with water and invert to dry on a paper
towel.
9. Calculate the concentration of RNA. Multiply the absorbance reading by 4000 (a
100 fold dilution of sample x 40 conversion factor) to get the concentration in
g/ml. Multiply this value by the sample volume (0.1-0.4 ml) to obtain the total
amount of RNA.
10. Calculate the volume that contains 5 g of RNA. Divide 5 g by the
concentration of RNA (g/ml) to find the total ml of RNA solution that you need
for the reverse transcription reaction.
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Reverse Transcription reaction for synthesis of first strand cDNA:
1. Prepare RNA/primer mixtures in sterile eppendorf tubes. Add up to 5 g of your
sample of total RNA to each tube. One of the tubes should have 1 l of control
RNA (from the kit) that is known to work well in the reverse transcription
reaction. Another tube should be set up as a ‘no reverse transcriptase control’.
This tube should get 5 l of your sample RNA but no enzyme. Add 1 l of 10
mM dNTP mix to each tube. Add 1 l of oligo (dT) to each tube. Bring the
volume of each tube to 10 l using sterile deionized water from the kit.
sample
RNA #1
RNA #2
RNA #2
Control RNA
RNA
5 g
5 g
5 g
1 l
dNTP
1 l
“
“
“
Oligo(dT)
1 l
“
“
“
H2 0
up to 10 l
“
“
7 l
2. Incubate each tube at 65C for 5 min, then place on ice for at least 1 min.
3. Prepare the ‘master mix’ solution containing all of the reverse transcriptase
reaction components. For each reaction tube you should add the following volume
of components:
10X reaction buffer….2 l for each tube
MgCl2……………….4 l “ “
DTT…………………2 l “ “
RNAse inhibitor……..1 l “ “
Prepare enough ‘master mix’ for n+1 tubes (if you have 4 tubes, prepare enough
for 5 tubes).
4. Add 9 l of the master nix to each RNA/primer tube, mix gently, and collect
contents at bottom of tube by brief centrifugation.
5. Incubate the tubes at 42C for 2 min.
6. Add 1 l (50 units) of superscript II reverse transcriptase to each tube except the
one designated as a ‘no reverse transcriptase control’. Mix the tubes by pipetting
gently, and incubate at 42C for 50 min to allow the reverse transcription to
occur.
7. Terminate the reaction by transferring the tubes to a 65C water bath for 15 min.
After heating, chill the tubes in ice water for 2 min.
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8. Centrifuge the tubes briefly to collect the reaction components at the bottom of
the tube. Add 1 l of RNAse H to each tube and incubate for 20 min at 37C to
digest any remaining RNA. All molecules that remain in the tube should be single
stranded cDNA.
Perform PCR on the RNA-DNA hybrids:
1. Assemble 7 thin wall PCR tubes. Set up your reaction tubes for PCR in the
following order:
Tube 1: use 2 l of your high molecular weight cell DNA and primers for P450
Tube 2: use 2 l of cDNA and primers for cytochrome P450
Tube 3: use 2 l of cDNA and primers for cytochrome occludin
Tube 4: use 2 l of cDNA and primers for integrin beta 4
Tube 5: use 2 l of cDNA and primers for HGF
Tube 6: use 2 l of the no reverse transcriptase control plus HGF primers
Tube 7: use 2 l of control RNA reaction and control primers from kit
2. After adding DNAs, cDNAs, and primers, add 45 l of PCR supermix to each
tube and mix contents briefly by pipetting. Place tubes in the thermocycler.
3. Turn the thermocycler on and select program 002. This is designed to denature at
94C, to anneal at 55C, and to extend at 72C. There are 30 cycles in this
program. Push the start button and allow the PCR to proceed.
Check products of the reverse transcriptase and the reverse transcriptase PCR
reactions:
1. Microwave 0.8 % agarose in 1X TAE buffer containing ethidium bromide. Allow
to cool to 65C and pour 2 mini gels (5-6 mm thickness). Allow the gels to
polymerize for 30 min.
2. Add 10 l of each reverse transcriptase reaction product to 3 l of 6X gel loading
buffer (blue juice) and mix well. Load into the first mini gel . Load 10 l of 1 kb
ladder into the 5th lane.
3. Add 20 l of reaction product from each PCR reaction into an eppendorf tube.
Add 4 l of blue juice and mix well. Load 10 l of 100 base pair molecular
weight ladder into the last lane.
4. Run the gels at 90 volts for 1 hour.
5. Photograph gels and place the picture in your lab book.
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