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Molecular
Biology Protocols Mount
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11. Analysis of Gene Expression by Real-Time
Quantitative PCR
Real-time PCR is similar
to conventional PCR except that the formation of amplification products is monitored
at each cycle, permitting the determination of the threshold cycle (Ct)
at which the product first begins to appear.
The simplest approach is to amplify the target cDNA in the presence of
the dye SYBR Green, which upon binding to double-stranded DNA produces an
intense fluorescent signal. A dilution
series will produce a family of curves representing successive dilutions. The abundance of cDNA template determines the
Ct value, with high template availability producing a lower Ct:
If the Ct data are plotted as a
function of template availability, the following relationship results:
Quantitative PCR measures the abundance of a
specific cDNA in a mixture of cDNAs prepared from tissue mRNA, and thus permits
the analysis of the level of expression of a specific gene.
Preparation
of RNA and cDNA
High quality RNA is
essential for reproducible results with quantitative PCR. We have found that the Promega
RNAgents Total RNA kit produces a consistently high
quality RNA preparation that is suitable for quantitative PCR. Several of the rapid protocols available do
not produce such consistency.
Measurement of RNA concentrations by UV absorbance at 260 nm or by
analysis with the Agilent 2100 Bioanalyzer is necessary to insure equal amounts
(2 μg) of total RNA for each reverse
transcription reaction. We find that the
Invitrogen Superscript First-Strand Synthesis kit produces consistent and
robust cDNA products for QPCR.
Stratagene
Brilliant SYBR Green QPCR System
The Stratagene MX4000
Real-Time PCR instrument is a 96-well format machine with four optical channels
for measuring four different fluorescent dyes simultaneously. We will use two of those channels, one for
SYBR Green and one for a reference dye (ROX) to correct for volume and plate
location differences. The primers
selected for QPCR should be of the highest specificity for the chosen target,
producing a single band with no smears on conventional PCR. A standard curve should be generated, using a
cDNA containing substantial levels of the desired template. A dilution series of x1 (undiluted), x10,
x100, and x1000, done in triplicate, is generally sufficient.
1. Add 1 μl of
the Stratagene reference dye (ROX) to 500 μl
nuclease-free water, to make a 1:500 dilution.
Each reaction mixture
will include the following:
12.5 μl
2X Stratagene Brilliant SYBR Green QPCR Master Mix (includes buffer, DNA
polymerase, dNTPs, SYBR Green)
0.3 μl
Forward primer at 25 μmol l-1
0.3 μl
Reverse primer at 25 μmol l-1
0.38 μl
Reference dye (ROX) (1:500 dilution)
10.52 μl Nuclease-free water
1 μl cDNA template
Each template should be
analyzed in triplicate. Combine the
first five reagents in sufficient volume to supply each reaction plus one or
more extra. For example, for 96 reactions,
mix together in one 12-ml microcentrifuge tube:
1250 μl
2X Stratagene Master Mix
30 μl
Forward primer at 25 μmol l-1
30 μl
Reverse primer at 25 μmol l-1
38 μl
Reference dye (1:500 dilution)
1052 μl
Nuclease-free water
Mix the cocktail very
well, and pour a portion into a 2-ml tube for ease of pipetting. Dispense 24 μl
of the mixture into each Stratagene reaction tube, which is a
specially-manufactured 0.5-ml tube in an 8-tube strip with optically clear
caps. Be sure to use Stratagene reaction tubes (Cat. No. 410022) and caps
(Cat. No. 410024).
Tubes and caps from other sources may cause the instrument to jam,
necessitating a service call that would take the instrument out of service for
several days to a week or more. Mark
the “A” end of the strip lightly on the
side of the tube (1-12) to insure correct orientation throughout the
assay. (Do not mark the caps of
course.)
2. Carefully
measure 1 μl of the appropriate cDNA template into the 24 μl of reaction mixture. This pipetting step
is the most critical of all of the steps in the procedure and must be done with
great care. The amount of template you
add will determine the Ct value and thus the level of gene
expression. Consistency is
essential. The recommended pipette is
the Rainin L2 with GP-L10F tips.
3. Close the reaction tubes using the Stratagene
cap strips and thoroughly mix the cDNA template with the reaction mixture by
vigorous tapping on the side of the tube or vortexing. Briefly centrifuge the tubes in a 96-well
plate carrier, balancing the tubes in two plastic 96-well plates. Do not centrifuge in the metal tube racks
that Stratagene provides with the instrument.
4. Turn on the MX4000 using the switch in the
rear of the instrument and activate the MX4000 software on its computer. Select “SYBR Green with Dissociation
Curve”. (If you are running Taqman or Molecular Beacon assays, consult with the center
director.)
5. Open the reaction chamber of the MX4000 by
clicking on the “open door” icon and place the tube strips in the
sample carrier, with tube A1 at the far left corner. Check that each of the caps is tightly
sealed. Before going on to the next
step, BE SURE THAT YOU HAVE USED STRATAGENE TUBE STRIPS AND CAPS! Other tubes and caps are likely to cause a
mechanical jam, damaging the instrument and requiring a service call. Now close the reaction chamber door by
clicking on the “close door” icon.
6. Program Plate Setup by highlighting occupied
wells using the mouse. Select well type:
“Unknown”. Collect
fluorescence data for FAM (~SYBR Green) and indicate the reference dye as
ROX. If you have a series of standards, those may
be programmed into the setup as well, indicating the amount of the standard in
the appropriate window. You may also
indicate replicates if you wish, enabling the software to compute means.
7. Program Thermal Setup by entering numbers in
the appropriate box or by “grabbing” the appropriate line and
moving it to the desired value. (Or
import a setup from a previous run.) A
typical thermal profile is the following:
95oC for 15
min (to activate the DNA polymerase)
40 cycles of:
·
94oC
for 40 sec
·
45 or 55oC
for 40 sec (In general, use 45oC for degenerate primers and 55oC
for specific primers. Other annealing
temperatures may be used if they are known to produce optimum amplification.)
·
72oC
for 60 sec (Take
triplicate readings of fluorescence during this phase of the cycle.)
To generate a
dissociation curve after the amplification cycles are completed, program 95oC
for 1 min, then 82 cycles starting at 55oC and increasing by 0.5oC
per cycle. Add segments and edit as
necessary, taking fluorescence readings at each temperature.
8. Save the file, turn
on the lamp if is not already on, and start the assay. Check the appropriate box to turn off the
lamp after the assay is complete unless you plan a second run immediately.
Data
Analysis
1. Check the dissociation plots for the possible
presence of two or more amplification products (indicated by two or more peaks
in the –R’(T) plots. The ideal of course is one product (one
peak)!
2. For the amplification plots, analyze
fluorescence as “dRn” to generate Ct
values for all of the samples simultaneously.
Set the threshold at 0.009 to permit comparisons between different
runs. Request a text report showing only
the Well Number and the Ct values.
Export the text report to an Excel spreadsheet. For the standard dilution series, plot Ct
against the log10 of the volume of template (in μl). Generate a slope and y-intercept for these
standards and use those values to convert Ct of the unknowns to a
relative expression value with respect to the standard. An Excel spreadsheet may be used for this
purpose. Graph your results with an
appropriate bar graph, including mean and standard error.