Establishing meaningful SYBR Green-based qPCR assays

SYBR Green qPCR with Standard Curve Protocol

Note from the Author:
This protocol is based on experiences using the StepOnePlus™ Real-Time PCR System (Applied Biosystems®).
It’s meant to help users generate meaningful and reliable qPCR results.

Procedure

Step 1: RNA purification

1. Use the appropriate commercially-available kit depending on the species and sample type (mammalian cells/tissue/blood, yeast, plant).
2. Follow the protocol carefully, being ultra cautious about contamination with RNAses. Do not touch anything with bare hands, change gloves often, cleaning benchtop and equipment with commercially-available RNAse removal spray (e.g. RNAse Away, Cat. nr 038184, MBP).
3. All plastic-wear (pipette tips, 1.5mL polypropylene tubes, etc.) should be purchased “RNAse-Free“. Do not autoclave plastic tubes prior to use.
4. It’s generally a good idea to have a good supply of RNAse-free water (Cat. nr R0013) before you start!

Commercially-available kits for various RNA purification applications:

• Mammalian cells and tissues:
  • Cultured Cell Total RNA Purification Kit (Cat. nr U0067)
  • MasterPure RNA Purification Kit (Cat. nr MCR85102)
  • Blood/Cultured Cell Total RNA Purification Kit (Cat. nr U0064)
  • Tissue Total RNA Purification Kit (Cat. nr U0066)
• For purifying very small quantities of RNA from very few mammalian cells:
  • ArrayPure Nano-scale RNA Purification Kit (Cat. nr MPS04050)
• For purifying only polyadenylated mRNA from mammalian cells/tissue:
• • BioMag SelectaPure mRNA System (Cat. nr 8MB4003K-1)
• Yeast:
  • MasterPure Yeast RNA Purification Kit (Cat. nr MPY03100)
• Plant:
  • MasterPure Plant RNA Purification Kit (Cat. nr MPR09100)
  • Plant Total RNA Purification Kit (Cat. nr U0065)

Step 2: RNA quantity and quality assessment

After the final step of RNA purification using one of the kits above:

1. Take a small aliquot (5-10ul) of RNA for quality testing.
2. Transfer this aliquot to a new “RNAse-free” tube. Store the rest of the sample at -80C.
3. Determine the concentration (ng/ul) of your purified RNA using a nanodrop machine or equivalent.

You will have to blank the machine with the same solution (generally RNAse-free water) you used to elute the RNA in the final step of the RNA purification procedure.
In general, you will be interested mostly in the concentration from the nanodrop machine, but some of the other values can give you some information about contamination with protein or solvents:

• A 260nm-to-280nm ratio of at least 2.0 is considered to be relatively free of protein and solvents.
• A 260 nm-to-230 nm ratio is generally from 2.0 to 2.2 and lower values indicate the presence of carbohydrates or solvents.

For quality testing, you can either run an RNA gel, using RNA Marker High Easy (Cat. nr R0004) as a molecular weight guide or use an Agilent Bioanalyzer or equivalent machine to quantitatively calculate the ratio between 28S and 18S RNA to determine if there has been degradation (or RNAse contamination).

Figure 1:

RNA Gel Results showing 28S:18S ratios.

Image kindly provided by Tebubio’s laboratory, Moloecular Biology Service division.

Even if your image acquisition software does not allow calculation of band intensities, you can generate fairly good 28S:18S values using ImageJ software. To do this, open your image file with ImageJ, draw a rectangular box in an empty/black area and click Analyze-Measure. This is your background value you will subtract from the 28S and 18S values. Then drag the box to the 28S band, click Analyze-Measure and then repeat for the 18S band (Figure 2).

Figure 2.​

ImageJ quantification of 28S:18S ratio

First measure the background (left), then measure the 28S band (middle), then measure the 18S band (right). Subtract the background from the 28S and 18S values and calculate the ratio.

You can also use the ImageJ Analyze-Plot Profile Function to generate a histogram showing the relative 28S and 18S quantities and degradation.

Figure 3.

ImageJ Profile Plot assessing RNA quality.

Using this RNA gel image found on the internet, the ImageJ (Analyze-Profile Plot Function) was used to draw a histogram, showing a low 28S:18S ratio and significant smearing below the 18S band, indicating poor RNA quality.

If you are using the Agilent Bioanalyzer, you will rely on both the 28S:18S ratio and their RNA Integrity Number (RIN) described here.

The Agilent Bioanalyzer uses a microfluidics approach to assess the quality of the RNA  (figure 4 for typical results), but the principle is reasonable similar to that described above. Using a microfluidics approach, the Agilent Bioanalyzer determines the relative abundances of 28S and 18S RNA as well as the degree of degradation. The software creates digital plots that resemble traditional RNA gels (left) and calculates the RNA Integrity Number (RIN) based on the plotted histogram (right).

Figure 4.

Sample Agilent Bioanalyzer Results.

Note: For many applications it is best to use only RNA with 28S:18S>1.5 and RIN>8.0.

Step 3: First Strand Synthesis (Production of cDNA)

Starting with the RNA you have been storing at -80C (see Step 2), follow the protocol from the cDNA synthesis kit of your choice. These kits generally require anywhere from 1ng to 5ug of RNA. Refer to the nanodrop concentrations you obtained in Step 2 and perform First Strand Synthesis on equal quantities (ng) of RNA for all of your samples. You will need to equalize the volumes with RNAse-free water.

Commercially-available kits for cDNA synthesis:

• • All-in-One First-Strand cDNA Synthesis Kit (Cat. nr AORT-0020)
  • TATAA GrandScript RT Kit (Cat. nr A103a)
  • Reverse Transcriptase Core Kit 300 (Cat. nr RT-RTCK-03)
  • MMLV Reverse Transcriptase (Cat. nr ENZ-310)
  • MonsterScript RT KIT (Cat. nr MS040910)

After completing the protocol associated with the kit you have chosen, it is recommended to clean-up the cDNA with a commercially available kit (e.g. Qiagen’s Qiaquick PCR clean-up or MACHEREY-NAGEL’s NucleoSpin Gel and PCR Clean-up) to remove residual oligomers and reverse transcriptase.
Dilute your cDNA samples at least 1:5 in RNAse free water, take a small aliquot (10ul-20ul) from each one, and store the remaining cDNA samples at -20C or -80C until you need them.

Step 4: Testing your qPCR Primers

Whether you purchased commercially-available qPCR primers or created your own using our protocol Design your own qPCR Primers (see technical tips in the Pasteur Alternative Careers Blog), it is important to test the primers using your samples, your SYBR Green Master Mix, and your qPCR machine to be certain that you are amplifying a single amplicon and that it is the proper sequence for the mRNA you are trying to measure.

Choose one of these commercially-available SYBR Green or MESA Green Master Mixes:

• • All-in-One qPCR Mix (Cat. nr QP005)
  • Takyon ROX SYBR MasterMix blue dTTP (Cat. nr UF-RSMT-B0701)
  • MESA GREEN qPCR MasterMix Plus (Cat. nr RT-SY2X-03+WOU)

Note: Always confirm that the qPCR master mix is compatible with the qPCR machine that you will be using!

Procedure for preparing the qPCR plate for initial testing of qPCR primers:

1. Starting with the 10-20ul small aliquots you took at the end of Step 3 above, create a single pool of all your cDNA samples for this qPCR Primer testing purpose. Make a dilution series (1:5) from this pool so that you will be able to see where your Ct values are in the measurable range. It should be sufficient to have the following concentrations: 1:1, 1:5, 1:25, 1:125.
2. The primer pair (Forward and Reverse) stock solution should be at 10uM concentration.
3. Prepare a master mix of SYBR Green Master Mix (2X) and primers (0.4uM).
4. Mix briefly and pipette 10ul into each well of the qPCR plate. To avoid bubbles, push the pipetteman plunger to the eject position prior to collecting the mixture. The pipette tip will now contain more than 10ul, but will only eject 10ul (and no bubbles) if you stop at the first resistance point while ejecting directly into the bottom of the well. Add 10ul to all of the wells that you will be using.
5. Use a separate pipette tip for each well, and add 10ul of sample (properly diluted with water) directly into the SYBR Green Master Mix containing primers. Mix by pipetting up and down one or two times. Do not use the eject function of the pipetteman for this step. Always also run a negative control (master mix, primers, and water) so that you can test for the presence, melting temperature, etc. of primer dimers.
6. Seal your plate with qPCR optical seals (cat. nr RT-OPSL-25) and start the qPCR run on your qPCR machine. If your machine’s software makes you choose, you will want to indicate that you are using SYBR Green reagents (not Taqman), that you want the long protocol (not the Fast protocol), that you are using a delta-delta Ct method, and that you want to include melting curve determination. If you are forced to choose a reference gene and a reference sample, choose any one since you are just testing your qPCR primers.
7. After the qPCR Run, use the software to evaluate the results, but save the plate at 4C for step 5.

Melting Curves (Testing specificity):
Using the qPCR machine’s software, look at the melting curves for each primer pair being tested to see if there is a single temperature peak (some examples of good and bad melting curves are depicted in figure 5):

Figure 5.

Melting curves.

Acceptable melting curves are those showing a melting curve (blue) distinct from the melting curve you get with the negative control (yellow).

A good primer pair will give a specific peak (left) while unacceptable primer pairs will yield products with less pure melting curves (middle and right).

Amplification Plots (Finding the Linear Range):
Next, using the qPCR machine’s software look at the amplification curves for each of the dilutions 1:1, 1:5, 1:25, 1:125 prepared above along with that of the negative control. Each 1 unit change in Ct should represent a 2-fold difference in starting material, so you should see a shifting towards higher Ct values with more dilute samples. What you need to look at here is the shape of the curve. Samples that are too concentrated may have an S-shaped amplification curve, while samples that are too dilute might have no curve at all or have very high Ct values.

Figure 6.
Amplification Curves.

Acceptable amplification curves are C-shaped (left). cDNA that is too concentrated may yield an S-shaped curve (right), however further dilution of the sample increase Ct values to the measurable range of 20-30.

Based on the results of this analysis, you will know to what degree (1:1, 1:5, 1:25, or 1:125) you will need to dilute your cDNA prior to using this primer pair to see these C-shaped curves when you perform Step 7.

Step 5: Sanger Sequencing of the Product and Generation of Standard Curve

Now starting with the completed/sealed qPCR plate that you have stored at 4C, purify the DNA with a commercially available kit (e.g. Qiagen’s Qiaquick PCR clean-up or MACHEREY-NAGEL’s NucleoSpin Gel and PCR Clean-up).
Determine the concentration (ng/ul) of your purified DNA using a nanodrop machine or equivalent. You will have to blank the machine with the same solution (generally DNAse-free water) you used to elute the DNA in the final step of the DNA purification procedure. Record this concentration, which will be very important for you to know the true abundance (ng) of PCR product you are using when you generate your standard curve. Send a small aliquot of the purified PCR product and either the forward or reverse primer to a company that performs Sanger sequencing as a service following carefully their instructions regarding sample and primer concentration. Costs are generally 5 EUR/sequence. When they send you the results confirm that the
amplified product is indeed the gene of interest. You can also look at the purified product on an EtBr-stained agarose DNA gel to be certain that there is a single band at the predicted molecular weight, but sequencing is generally sufficient.

To generate the standard curve dilute the sequenced PCR product from above1:100 in 1X TE buffer, pH 7.5 (Cat. nr MB-006) with 10ug/ml Sheared Salmon Sperm DNA (Cat. nr MB-103-0025). For example, add 10ul of the purified PCR product to 990ul buffer. Then perform a 1:10 dilution series in the same buffer to prepare the standards: 1:103, 1:104, 1:105, 1:106, and 1:107.
It is recommended to make at least 500ul of each standard and store them at -20C or -80C for future use.

Note: When handling these amplified PCR products it is very likely that you will contaminate your pipetman. Use filter tips and consider borrowing your neighbour’s pipetman for this step.

Step 6: Assessing Efficiency of your qPCR

Now that you have generated your standard curve, you can test these samples using your qPCR machine. Using precisely the same procedure as in step 4, perform qPCR on your standards, each one in triplicate. Your machine may allow you to enter the true quantities (ng) of each standard, which you should know based on the nanodrop results in step 5.

As you evaluate your standards ( 1:103, 1:104, 1:105, 1:106, and 1:107), you may find that some are not in line with the others because they are too dilute or too concentrated. You can eliminate these from the analysis find the best 3 or 4 standards for your assay. In general, standards do not suffer from the S-shaped curve phenomenon described in Figure 6, and a linear range can even extend into Ct values around 10 (figure 7).

Figure 7.

qPCR Standard Curves.

Acceptable standard curves will have an R2 near 1.0 and an efficiency of 100% or less.

If after discarding these outliers your standard curve still has an unacceptable R2 value, you will likely need to re-make the standard curve, taking more care to mix each dilution well prior to moving on to the next dilution.

An efficiency with greater than 100% may indicated more significant problems, however, and may require you to design a new primer pair.

Step 7: Performing qPCR on your Samples

You are now ready to perform qPCR on the cDNA samples you have been storing frozen since the end of Step 3.

Procedure for preparing the qPCR plate for qPCR on your samples:

1. Starting with the 1:5 diluted cDNA you froze at the end of Step 3 above, dilute all samples appropriately (1:1, 1:5, 1:25, or 1:125) according to the results you obtained in Step 4. You can pre-dilute all of your remaining cDNA to this dilution, but you should be aware that some primer pairs will give significantly different amplification curves than others. If you’re working with a very abundant transcript (e.g. that of GAPDH), for example, you might dilute your cDNA too much so that you won’t be able to detect less abundant transcripts.
2. The primer pair (Forward and Reverse) stock solution should be at 10uM concentration.
3. Prepare a master mix of SYBR Green Master Mix (2X) and primers (0.4uM).
4. Mix briefly and pipette 10ul into each well of the qPCR plate. To avoid bubbles, push the pipetteman plunger to the eject position prior to collecting the mixture. The pipette tip will now contain more than 10ul, but will only eject 10ul (and no bubbles) if you stop at the first resistance point while ejecting directly into the bottom of the well. Add 10ul to all of the wells that you will be using.
5. Use a separate pipette tip for each well, and add 10ul of sample (properly diluted with water) directly into the SYBR Green Master Mix containing primers. Mix by pipetting up and down one or two times. Do not use the ejectfunction of the pipetteman for this step. Samples should be analyzed in at least triplicate. Always also run a negative control (master mix, primers, and water) so that you can test for the presence, melting temperature, etc. of primer dimers. On the same plate also load your standard curve in duplicate or triplicate.
6. Seal your plate with qPCR optical seals (Cat. nr RT-OPSL-25) and start the qPCR run on your qPCR machine. If your machine’s software makes you choose, you will want to indicate that you are using SYBR Green reagents (not Taqman), that you want the long protocol (not the Fast protocol), that you are using a standard curve method, and that you want to include melting curve determination.

The qPCR machine will then produce meaningful quantitative PCR results and tell you the true quantities (ng) of your transcript in each sample. You will be certain of the sequence, specificity, and linearity of the assay.

Legal notes:
StepOnePlus is a registered trademark of of Applied Biosystems. NanoDrop is a registered trademark of NanoDrop Technologies, Inc. TaqMan is a trademark of Roche Molecular Systems, Inc. RNase AWAY is a registered trademark of Molecular Bio-Products, Inc. Qiaquick is a registered trademark of the QIAGEN Group. NucleoSpin® is a registered trademark of MACHEREY-NAGEL. SYBR is a registered trademark of Molecular Probes, Inc. Other parties’ trademarks are the property of their respective owners and should be treated as such.