TITLE: Quantitative (q)PCR and Differential Expression Analysis
AUTHORS: Jeffrey Lewis, Amanda Scholes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A generalizable protocol for measuring relative changes in gene expression via qPCR (cDNA synthesis, qPCR primer optimization, and qPCR analysis), with specific optimization for the budding yeast </span><span style = "font-style:italic;">Saccharomyces cerevisiae.</span></div></div>

[STEPS]
?. [qPCR Primer Design]
1. Primer Design
We use Primer3 to design primers: http://biotools.umassmed.edu/bioapps/primer3_www.cgiPrimers are designed to have a Tm as close to 58°C as possible. This helps to ensure that primer annealing will be similar for all reactions. Primers should amplify a product of ~100 - 200 bp. Longer products can have reduced PCR efficiency, and are more susceptible to differences in RNA degradation levels. For gene expression analysis, primers are designed within the 3' end of an ORF. This mitigates against partial cDNA synthensis, especially if only oligo-dT is used for cDNA generation.Primers are designed to have a Tm as close to 58°C as possible. This helps to ensure that primer annealing will be similar for all reactions. Primers should amplify a product of ~100 - 200 bp. Longer products can have reduced PCR efficiency, and are more susceptible to differences in RNA degradation levels. For gene expression analysis, primers are designed within the 3' end of an ORF. This mitigates against partial cDNA synthensis, especially if only oligo-dT is used for cDNA generation.Primers should amplify a product of ~100 - 200 bp. Longer products can have reduced PCR efficiency, and are more susceptible to differences in RNA degradation levels. For gene expression analysis, primers are designed within the 3' end of an ORF. This mitigates against partial cDNA synthensis, especially if only oligo-dT is used for cDNA generation.
?. [cDNA Synthesis]
1. Prepare RNA/Primer mixture:        10 μg total RNA          3 μg anchored oligo-dT (T20VN)          3 μg random hexamer (optional)2. Adjust volume to 7 μl with RNase-Free TE.3.  Denature RNA/Primer mixture at 70°C for 10 min, then immediately chill on ice for > 2 min.4. For each reaction, prepare the Reverse Transcriptase Master Mix        3.0 μl 5x Superscript Buffer        1.5 μl 0.1M DTT        3.0 μl 10x RNase-Free dNTPs (2.5 mM stock solution, final concentration of 250 μM)        0.5 μl Superscript III RT5. Add 8 μl Reverse Transcriptase Master Mix to the denatured RNA and incubate on bench (25°C) for 7 min.6. Incubate at 50°C for 2 hrs in a thermal cycler.
If you are looking solely at poly-adenylated transcripts (e.g. eukaryotic mRNA) only oligo-dT is needed. Random hexamer is necessary to generate cDNA from non-coding RNAs and prokaryotic mRNAs. Note: cDNA synthesis using random hexamer generates more total cDNA, but with a smaller overall fraction of mRNAs represented, and you may need to add more cDNA to your qPCR reactions.
When making the Master Mix, prepare enough for the number of samples plus one (e.g. 25 reaction volumes for 24 samples). This ensures that there will be enough MasterMix for all aliquots.
If doing no reverse transcriptase (RT) control reactions, prepare a no RT control Master Mix by substituting Superscript III with an equal volume of RNase-Free water.
?. [RNA Hydrolysis and Cleanup]
1. To each reaction, add        10 μl 1 M NaOH        10 μl 0.5 M EDTA, pH 8.02. Incubate at 65°C for 15 min. 3. Immediately proceed to cDNA cleanup.4. Analyze concentration via Nanodrop or Qubit.
We use the Zymo Clean and concentrator (D4014), according to the manufacturer's instructions. We also add the eluate back onto the column and repeat the elution to increase cDNA yield.
?. [Quantitative PCR]
1. Design qPCR plate layout using only the interior 60 wells. 2. Dilute each primer to 10 μM. 3. Dilute each cDNA sample 5-fold (e.g. if you are using 1 ng cDNA per reaction, dilute the cDNA to 0.2 ng / μl).4. Prepare a qPCR Mastermix for each primer pair (N+1). For each reaction you will add the following amounts:        0.4 μl Forward Primer (final concentration 200 nM)        0.4 μl Reverse Primer (final concentration 200 nM)        4.2 μl water        10 μl Maxima 2X SYBR Green Mix (1x final)5. Pipette 5 μl diluted cDNA to the bottom of the appropriate wells of a BioRad RT-PCR plate. Be careful not to touch the tips to the side of the wells. Use a single pipette tip for pipetting the same cDNA sample into multiple wells.6. Pipette 15 μl qPCR Mastermix to the side of the appropriate wells. Use a single tip for each mastermix. Change tips when switching to a new mastermix (different primers). Be careful to only touch the side of the well so as to not contaminate the pipet tip with cDNA already in wells. 7. Seal qPCR plate with clear Bio-Rad sealing membrane; use roller if applicable. 8. Briefly spin the plate in a microplate centrifuge. This ensures all the qPCR mastermix reaches the cDNA in the bottom of the well. 9. Reaction conditions will be primer specific. Based on a primer Tm of 58°C we use the following cycling conditions:        95°C x 3 min        -----------------------------------        95°C x 15 sec        55°C x 1 min        40 cycles        -----------------------------------        Melt curve analysis (55°C - 95°C in 5 sec steps; default instrument settings)10: Use the following instrument settings:            Cq determination mode  = regression            Baseline settings = Baseline subtracted by curve fit with no fluorescence drift corrected            Analysis mode = Fluoropore
Do not use the outer edge wells of the PCR plate for your samples (no RT controls are okay), because uneven heating and/or evaporation increases variability. That means there are 60 usable wells for samples per PCR plate.
When making cDNA dilutions, prepare a large enough volume so that you are pipetting no less that 2 μl of cDNA. Pipetting volumes smaller than 2 μl will decrease the accuracy of your dilutions. (E.g. 2 µl (1 ng / µl) cDNA to 8 µl of water for a 0.2 ng / μl dilution).
Melt curve analysis allows us to check for co-amplification of non-specific products. More than one peak can indicate more than one PCR product, which should be verified via gel elecrophoresis on those samples.
?. [Validating PCR Primer Specificity]
1. To validate specificity of the primers, perform PCR on genomic DNA template from both the wild-type strain and a deletion strain for the gene of interest. Use the same polymerase (e.g. Taq) and cycling conditions as you would for qPCR:        95°C x 3 min        -----------------------------------        95°C x 15 sec        55°C x 1 min        30 cycles        -----------------------------------2. Perform gel electrophoresis (2% agarose, ~120V for 30-60 min) with the expectation of a single band (100 - 200 bp) in the wild-type and no band in the negative control (deletion strain).
You should see a single band for the wild-type control and no band for the negative control. If you observe a band in the negative-control sample and/or multiple bands in your wild-type sample, the primers are likely annealing to other locations in the genome and should be redesigned.
?. [Validating qPCR Primer Efficiency ]
1. Dilute cDNA to a range of concentrations: 0.01 ng, 0.05 ng, 0.1 ng, 1 ng, 5 ng, 10 ng, 25 ng and 50 ng (can adjust higher or lower if necessary)2. Perform qPCR (Step 6) on new primers (plus the control gene) on the range of cDNA concentrations.3. Determine your Ct (aka Cq on BioRad qPCR machines) values for your test gene and control gene at each cDNA concentration.4. Log10 transform your cDNA concentrations (e.g. log10 of 0.01 = -2 and log10 of 50 = 1.69)5. Plot the data (e.g. in GraphPad Prism or Excel) with Ct-values on the y-axis and log10-transformed cDNA concentrations on the x-axis.Carefully examine that plot for cDNA concentrations that fall out of the linear range of the assay. If necessary repeat the cDNA dilutions (step 1) to exclude cDNA concentrations outside of the linear range, and to include more cDNA concentrations that fall within that range.        6. Perform linear regression analysis to calculate the slope and r2 of the line.The r2 of the regression is typically close to 0.99. 6. Perform linear regression analysis to calculate the slope and r2 of the line.7. Calculate efficiency of each primer set using the equation: Efficiency(%) = (10(-1/slope) - 1) x 1008. Generally, primer-pair efficiencies >90% are acceptable (closer to 100% is best), and all primer pairs (experimental and control genes) should have efficiencies within 5% of each other.
Use a cDNA sample where you are certain that your gene of interest is being expressed. We standardly use a wild-type strain grown under "control" conditions.Note: the recommendations for concentrations here assume that cDNA was generated using oligo-dT and not random hexamer. Higher concentrations of cDNA may be necessary if using random hexamer (see Step 4).
Example:         Slope of regression line: -3.353        (10(-1/-3.353) - 1) x 100 = 98.7%
Efficiencies higher than 100% could be do to pipetting errors, or due to the presence of polymerase inhibitors (most likely in the cDNA).
?. [qPCR Data Analysis]
ΔΔCt Analysis1. Normalize the Ct values by subtracting the Ct for your control gene from the Ct of the target gene.            ΔCt (sample) = Ct (target gene) - Ct (control gene)2. Calculate the ΔΔCt using the normalized values from step 1.            ΔΔCt = ΔCt (treated sample) - ΔCt (untreated sample)Therefore:            ΔΔCt = [Ct(target gene, treated) - Ct(control gene, treated)] - [Ct(target gene, untreated) - Ct(control gene,         untreated)]
The critical threshold cycle (Ct - also labelled as Cq on Bio-Rad instruments) is the cycle number where the fluorescence signal crosses a set threshold. Differences in Ct values between samples can be used to measure relative transcript abundance.We use the ΔΔCt method to quantify difference in relative trancript abundance, as described by Livak and Schmittgen (Methods. 2001. 25(4): 402-408.). This method assumes that both target and control (reference) genes are amplified with efficiencies near 100% and within 5% of each other (i.e. Step 3).
To convert ΔΔCt to fold changes, use the following equation: 2(-ΔΔCt)
?. [Validating qPCR Primer Efficiency ]