2. Do qRT-PCR to obtain dilution curve and calculate PCR efficiency for each pair of primer
(1) Make 7 serial five-fold dilutions of cDNA (could be any cDNA sample, but it’s better to use the mixture of the target samples to reduce the difference caused by templates). The cDNA amount units of the templates could be the dilution values 1, 0.2, 0.004, 0.0008, and so on.
(2) Set up PCR reactions. Do 3 duplicated reactions for each diluted templates and NTC.
(3) Each reaction set up is the same as above.
(4) qRT-PCR conditions and instrument setup are also the same as above.
(5) Data analysis by using Mx4000 software
a. Check dissociation curve to make sure that every reaction just has single peak
b. Check amplification curve to make sure there is no product formed for NTC (no contamination)
c. Set up proper threshold to get the Ct values in the range of exponential amplification (usually 0.3 works fine)
d. Export Ct values to Excel file (The default format is “.CSV”)
(6) Data analysis by using Excel
a. Input the dilution values and calculate log values for them in the exported Excel file of Ct values
b. Select the log dilution values and Ct data
c. Draw an XY (scatter) plot on the work sheet with the log input amount as the X values and Ct as the Y values
d. Open the insert menu and select trendline to plot a line through the data point and select linear regression
e. Go to the Options page and select the boxes for display Equation on Chart and display R2 value on chart. If the R2 value is lower than 0.98, you need to redo the experiments or do not use the primers for further experiments or analysis
f. Calculate E values according the following equation (E = PCR efficiency + 1)
g. Save the file as “.xls” file
3. Do qRT-PCR for the above primers by using the samples you want to compare as templates separately
(1) Make a certain diluted templates for cDNA samples according to previous results (Don’t use original cDNA samples directly for qRT-PCR because the buffers left after RT reaction would affect the qRT-PCR results).
(2) Set up PCR reactions. Do at least 3 duplicated reactions for each sample of each pair of primers. Each reaction is set the same as above.
(3) “qRT-PCR conditions and instrument setup” are the same as above
(4) Data analysis by using Mx4000 software
a. Check dissociation curve to make sure that every reaction just has single peak
b. Set up proper threshold to get the Ct values in the range of exponential amplification (usually 0.3 works fine)
c. Export Ct values to Excel file (The default format is “.CSV”)
d. Calculate the fold-change of each gene for the two samples and normalize them by using the spiking human gene as reference
Note: “control-sample” could be “sample 1-sample 2”.
(5) Data analysis to determine the difference: If you have enough biological replicates for the real time PCR, consult Dr. Dan Nettleton for statistical analysis. If you don’t, the following is a way to calculate the lower and upper limits of foldchanges. One way to determine the difference is (lower limit, upper limit) does not span 1.
MAIN REAGENTS AND EQUIPMENTS
• Oligonucleotide Primers. Gene specific primers are retrieved from Primer3. These primers are
ordered from IDT. Primer purification: standard desalting.
• SuperScript First-Strand Synthesis System for RT (Invitrogen).
• SYBR Green PCR master mix (Applied Biosystems, cat log: 4309155).
• 96-well plates (Stratagene, cat.: 410021)
• 8-tube strip tubes (Stratagene, cat.: 410022)
• Optical strip caps (Stratagene, cat.: 410024)
• 3% Agarose Gel.
• Agarose gel electrophoresis apparatus.
TIPS AND NOTES
1. If using different instrument or fluorescent dye, the protocol needs to be modified.
2. Find an adjusted pipette and keep it for the qRT-PCR set up.
3. Eliminate bubble before running PCR because it will affect the final Ct results.
4. If the initial set of 200nM primer doesn’t work, you can try to optimize the primer concentration.
5. Make mixtures for PCR setup to reduce the differences among reactions.
6. Before determining the reference gene, select several housekeeping genes and 18S rRNA to do some preliminary experiments.
7. Because the RNA I obtained is amplified RNA, I did the RT with random hexamer. If starting RNA is total RNA, it’s better to use polydT primer and modify the procedures.
8. If the dissociation curve gives multiple peaks (even though some are very short or weak), don’t use the primers for further experiments. Because it either means there is primer dimmers or nonspecific products. If it is only the primer dimmer, then it may be fixed by optimizing primer concentration. If the curve contains very ambiguous peaks, it is also possible that there are nonspecific products. To use these kinds of primers, it’s better to do sequencing before further
experiments. For example, there is more than one product by sequencing for the qRT-PCR amplicons, which has the dissociation curve as showed in the following figure.
