LSUHSC-S Genomic Research Core Facility Real-Time PCR Guidelines

The purpose of this document is to provide an introduction to real-time PCR. The information presented here is only meant to be a guide. It is highly recommended that you research this technology by querying online technical resources and scientific publications prior to beginning your first experiment.

Real-Time PCR/Quantitative PCR (qPCR) uses the same principle of amplification as endpoint PCR; however, the process is monitored in “real-time”. The signal is detected at each cycle of amplification.

Quantitative reverse transcription PCR (RT-qPCR) is used when the starting material is RNA. Total RNA is first transcribed into cDNA. The cDNA is then used as a template for the qPCR reaction.

- A typical real-time reaction consists of:o Increasing the temperature to 95°C to melt (denature) the DNA into single strands.o Lowering the temperature to ~60°C to allow the primers to bind (anneal) to your gene

of interest. o Increasing the temperature to 72°C to allow the bound polymerase to copy the DNA

strand more efficiently (extension). Most current protocols do not include this step, as this function can be performed in the previous step as well.

o Cycling the temperatures 40 times, generating an exponential amplification. One copy becomes 2 copies, 2 copies become 4 copies, 4 copies become 8 copies and so on until many copies are created.

- Detection is enabled by the inclusion of a fluorescent reporter molecule in each reaction. o DNA-binding dyes (SYBR Green Chemistry)

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o Fluorescently-labeled sequence-specific probes (TaqMan)

Note: Input for qPCR is not limited to cDNA and can include genomic DNA from any source as well as plasmid DNA. From here out, this guide specifically refers to cDNA as the input.

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Typical Reverse Transcription Quantitative Real-Time PCR Steps

Step 1: RNA Extraction and Quantification

Step 2: Assessment of RNA Integrity and Purity

Step 3: Primer Design

Step 4: cDNA Synthesis: Total RNA to cDNA

Step 5: Determination of Primer Specificity

Step 6: Determination of Sensitivity (Dynamic Range) and Efficiency by Standard Curve

Step 7: Real-Time PCR Reaction Setup

Step 8: Data Analysis

Step 1: RNA Extraction and Quantification

- Use an extraction method that produces high quality RNA. Recommendations include using a column-based Qiagen extraction kit or a TRIzol reagent (phenol:chloroform method); although, any method that yields high quality and pure RNA is acceptable.

- If you have significant genomic DNA contamination and must DNase-treat your RNA, use an RNase-free DNase. Keep in mind that inactivation of the enzyme usually requires heat and EDTA. EDTA can inhibit downstream applications such as reverse-transcription. If you must DNase-treat your RNA, check on alternative protocols that do not require EDTA, or make sure that your cDNA is sufficiently diluted before using it. Check out QIAGEN’s DNase Max Kit.

- Quantify your RNA on a nanodrop, spectrophotometer or fluorometer. Keep in mind that a nanodrop and spectrophotometer will detect anything that fluoresces at the wavelength being used to quantitate your RNA, including, but not limited to, genomic DNA and phenol. This will cause over-estimation of your concentrations.

Step 2: Assessment of RNA Integrity and Purity

- Run your RNA on an agarose gel or an Agilent Bioanalyzer to determine RNA integrity. The ratio of 28s to 18s rRNA should be close to 2.0 and the 28s and 18s bands should be sharp with no smearing in between them. If running the samples on an Agilent system, the RNA Integrity Number (RIN) should be at least 8.0 and should be consistent among samples you are comparing.

- Purity should be assessed by looking at the ratio of absorbance at 260 nm and 280 nm (A260/A280). A ratio around 2.0 is acceptable. If this ratio is considerably lower, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. Very low RNA concentrations will also result in low A260/A280 ratios.

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- Purity should also be assessed by looking at the ratio of absorbance at 260 nm and 230 nm (A260/A230). The A260/A230 value is often higher than the respective A260/A280 value and is expected to be anywhere from ~2.0 to 2.2. It this ratio is considerably lower, it may indicate the presence of contaminants that absorb at 230 nm, such as carbohydrates, phenol or TRIzol that has carried over from the isolation. Guanidine isothiocynate, which is used in Qiagen buffers, will absorb at ~260 nm.

Step 3: Design Primers

- Primer design is an important consideration for RT-qPCR. Primers should be highly specific to your sequence of interest. Blast your primers to check for specificity.

- Check primers for secondary structures, such as self-dimers and hairpins. As a general rule, the ΔG value for these structures should be more positive than -9.0 kcal/mole.

- Order validated pre-developed primers from places like IDT, when possible.- If designing your own primers, use a software specific for qPCR and the chemistry (SYBR Green

or TaqMan) you are using. Beacon Designer is available in the Core Computer Lab and the NCBI tool Primer BLAST can be accessed online.

o It is good practice to design and order several pairs for each sequence of interest. Test them and choose the best one.

o If you are having trouble finding the accession number that correlates to your gene, go to the IDT pre-developed assay page, query for your gene and retrieve the accession number from the returned results.

- When possible, design your primers to span an exon-exon junction. This design reduces the risk of false positives from amplification of any contaminating genomic DNA, since the intron-containing genomic DNA sequence will not be amplified.

- Consider the following when designing primers for SYBR Green Chemistry:o PCR product/amplicon size (80 to 150 is optimal).o The optimal melting temperature (Tm) of your primers is 60 to 64˚C. Ideally, the melting

temperatures of the 2 primers should not differ by more than 2˚C so that both primers bind simultaneously and efficiently amplify the product.

o The annealing temperature (Ta) is dependent upon the length and composition of your primers. In general, this temperature should not be more than 5˚C below the Tm of

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your primers. If the Ta is too low, one or both primers may anneal to sequences other than your intended target, leading to nonspecific PCR amplification. If the Ta is too high, you may see a reduction in reaction efficiency, as the likelihood of primer annealing is reduced.

o GC content should be between 35-65% with an ideal content of 50%. Primer sequences should not contain regions of 4 or more consecutive G residues.

- For more information on primer design or probe and primer design, visit IDTAhttp://www.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2013/10/21/designing-pcr-primers-and-probes

Step 4: cDNA Synthesis – Total RNA to cDNA

- Reverse transcribe your total RNA into cDNA. Kits are available that include pre-made master mixes.

- Don’t forget to include an RT- control! It is virtually impossible to eliminate genomic DNA from RNA preparations; therefore, it is important to include a minus-reverse transcriptase control (RT-) in your experiment. This is a mock reverse transcription containing all RT-PCR reagents except for the reverse transcriptase. If a product is seen in the subsequent qPCR reaction, it most likely indicates that contaminating DNA is present in the sample.

Step 5: Perform Endpoint PCR to Assess Primer Specificity

- Every set of primers should be optimized by running your PCR product on a gel or an Agilent Bioanalzyer to determine size and the presence of primer dimers or nonspecific products.

Step 6: Determine Sensitivity (Dynamic Range) and Efficiency

If a PCR amplicon doubles in quantity during the geometric phase of its PCR amplification, then the PCR assay is said to have 100% efficiency. This is a measure of the overall performance of a real-time PCR assay.

A dilution series of your template (standard curve) should be run in a real-time reaction to check the efficiency of your primers. For real-time PCR, a slope between -3.1 and -3.6 is acceptable.

Dynamic range is the minimum and maximum sample amount that may be run in the assay system to produce accurate and precise data.

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Use a serial dilution process to maximize standard curve quality.o Use a minimum of 5 different concentrations or “points” within approximately a 3-log

range (a 5-fold or 10-fold dilution factor is usually sufficient to achieve this range). Standard curves are plotted log quantity units on the x-axis and Cq/Ct values on the y-axis. The

Bio-Rad CFX and ABI 7900HT software will plot these curves if the plate template is set up and labeled correctly.

The software calculates the efficiency, the correlation, and the slope for you.

o The efficiency should be close to 100% (90% to 110% is usually deemed acceptable). A 100% efficient reaction indicates that the PCR amplicon is doubling with each

PCR cycle. The efficiencies of the primers used within your comparison experiment should

be equivalent.o A correlation (R2) of > 0.99 is recommended.o Ideally, the data points will generate parallel lines with negative slopes near -3.3 (-3.1 to

-3.6). Keep in mind that pipette calibration error can negatively impact the slope, but should not impact the correlation.

Problems uncovered should be resolved before you proceed with your experiment. For SYBR Green assays, each gene should be tested, because they may have unique tendencies

to produce non-target products as a function of sample or target amount, which would limit sensitivity and dynamic range.

Samples should be derived from the same source as your other samples, e.g., use a sample from blood when all other samples are from blood.

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o Samples should be extracted or lysed using the same methods as your other samples.o Samples should be non-precious.o The target and normalizer should be present in adequate abundance (to “survive” the

dilution process). Running standard curves on all samples is not necessary.

o Running even one representative sample as a standard curve should provide useful information, but running more than one sample is recommended when:

You have highly variable sample quality. You use different extraction methods or sample types (blood, vs. liver for

example). A previous dynamic range test revealed inhibitors.

Suggestions to Improve the Accuracy of your Dilution Curves:

1. Use well-calibrated pipets.2. Pipet accurately.3. Serially dilute standard material.4. Mix samples thoroughly after each dilution.5. Pipette higher volumes for better precision.6. Run triplicates of each dilution point in real-time.7. Prepare 10-fold dilutions and cover a minimum of 5 dilution points.8. Check the correlation value.9. Look for outliers and omit.10. If the curve has more than 1 or 2 outliers, or if the spacing between the points is irregular,

repeat the dilution curve.

Step 7: qPCR Reaction Setup

An example reaction is below. Please refer to your SYBR Green Mix for the appropriate reaction setup.

The Bio-Rad CFX96 Fast will accept total reaction volumes between 10 and 25 ul.

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Notes:

1. Make dilutions of your cDNA template to pipet at least 1 (2)ul of template into PCR reactions for accuracy.

2. Stock primers can be diluted to 20uM and added to your reaction at a final concentration of 300nM. This is a good starting place; however, optimizations may need to be performed if you are seeing the presence of significant primer-dimer formation.

3. Include template negative controls. For cDNA, an RT-negative control should be included for every set of primers for each new RNA extraction. A no-template control should also be included for every set of primers. Signal in RT-negative controls may be due to genomic DNA contamination.

4. Run samples at least in duplicate. If you are new to PCR you might want to run samples in triplicate.

5. Contamination is common in PCR. a. Be careful with your reagents. b. Use a PCR hood if possible. c. Clean pipettes before every use. d. Aliquot your water. e. Separate plasmid isolation from other molecular analysis (in different rooms if possible). f. Use barrier/filter tips.

6. Include a melt curve analysis if using SYBR Green, and check your melting curves after every run. SYBR Green dye will bind to any double-stranded DNA, including primer dimers,

contaminating DNA and non-specific PCR products. Melt curve analysis is used as a diagnostic tool to assess specificity.

As the temperature increases, the DNA starts to denature.

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The RT- control in conjunction with the melt curve analysis can determine if contaminating genomic DNA is present (IDT website):

A no template control (NTC) in conjunction with the melt curve analysis can determine if primer dimers or contamination are present (Kordo et al., Real-Time Polymerase Chain Reaction: Applications in Diagnostic Microbiology, IJMS, eISSN 2076-6327, 2012):

7. Check the MIQE Guidelines (http://www.rdml.org/clinchem.2008.112797v1.pdf) for publication of real time data.

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Step 8: Data Analysis – Relative Quantification (ΔΔCT Method)

Note: This guide only refers to the relative quantification method using the ΔΔCT calculation. If you are performing absolute quantification (standard curve method), please refer to an alternative source for information.

- The ΔΔCT analysis requires that the efficiency of primers be equivalent. Otherwise, a standard curve analysis should be performed.

- Relative quantification measures the relative change in mRNA expression levels.- It determines the changes in steady-state mRNA levels of a gene across multiple samples and

expresses it relative to the levels of another RNA.- You must select an endogenous control (housekeeping gene) to normalize input amounts.

o Select a gene with a stable pattern of expression across all conditions and sample types or tissues. Some endogenous controls may be accurate for certain sample types but not others.

- To determine the level of expression, the differences (Δ) between the quantification cycles (Cq) or threshold cycles (Ct) are measured. Cq and Ct can be used interchangeably. The designation depends solely upon the instrument and software you are using. From here out, we will just state Ct.

- Data is plotted as fluorescence signal versus cycle number.

The baseline is the initial cycles of PCR where there is little change in fluorescence signal. This defines the baseline for the amplification plot. In these cycles, the fluorescence background of the reaction is observed. This is subtracted when setting the baseline.

The threshold is the line whose intersection with the amplification plot defines the Ct. The threshold is the level of ΔRn used for the determination of the threshold cycle (Ct). This level is set above the baseline, but sufficiently lower to be within the exponential growth region of the amplification curve.

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- The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.

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Has the highest starting copy number

The second step is to subtract the experimental sample from the control sample. ΔCtLung – ΔCt Brain = ΔΔCt

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IMPORTANT CONTROLS

https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rtpcr-analysis/general-articles/ten-most-common-real-time-qrt-pcr-pitfalls.html#5

RUN A RT- CONTROL: It is virtually impossible to completely eliminate genomic DNA from RNA preparations. Therefore, it is important to include a minus-reverse transcriptase control ("No Amplification Control" or NAC) in qRT-PCR experiments. Typically, the NAC is a mock reverse transcription containing all the RT-PCR reagents, except the reverse transcriptase. If a product is seen in the NAC, it probably indicates that contaminating DNA is present in the sample

RUN A "No Template Control" (NTC): A No Template Control should be run to rule out cross contamination of reagents and surfaces. The NTC includes all of the RT-PCR reagents except the cDNA template. Typically the cDNA is simply substituted with nuclease-free water. No product should be synthesized in the NTC; if a product is amplified, it indicates that one or more of the RT-PCR reagents is contaminated with the amplicon or that you have primer-dimers.

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