Real-time PCR is a specialized technique that delivers far more information about your DNA or RNA than end-point PCR.
Essentially, real-time PCR is a way to visualize the amplification of specific DNA fragments as it is happening (in real time) and allows for the ability to quantify exactly how much DNA (or RNA) was in the original sample.
Fluorescent dyes (either SYBR Green or dye labeled DNA oligos) are mixed in the amplification reaction, fluoresce when they bind to DNA, and are measured by the machine during replication.
The amount of fluorescent signal measured directly correlates to the amount of product in the tube at that moment in time. Because of the sensitivity of fluorescent dyes, concentrations as low as picogram quantities of DNA (or as low as a single cell) can be accurately detected.
While it has become second nature in some labs, the technique does require a certain amount of technical finesse to get consistent and reliable data every time. Because the cost of real-time PCR kits is so much higher than standard PCR, getting every experiment right is critical.
The main bottlenecks people encounter when getting started with real-time PCR are contamination issues or inconsistency between replicates. Here are some simple but important pointers to help make sure you prevent the cause of many grey hairs and frustration at lab meetings.
1. Always mix the reagents well before use
The reagents contain dyes, nucleotides, and enzymes that may have settled while sitting in the freezer or refrigerator. Give the master mix a good mix before your start aliquoting into your plate or tubes to avoid uneven distribution of reagents between samples.
2. Store primers in a buffer to protect their stability
When your primers arrive, avoid resuspending the master stock in water. The pH of water can be low (especially if it is DEPC treated) and this will be damaging to DNA over time.
Use a buffered solution at neutral pH to protect from acid hydrolysis. EDTA (1 mM) in the master stock is also a good idea to protect against DNases and when you dilute the primers for working stocks, the EDTA will be sufficiently dilute that it will not interfere with Taq activity.
3. Aliquot the primers to avoid excessive freeze/thawing (and contamination)
Once you have a master stock (usually 100-200 mM), you will want to make some working stocks so that you are not continually freeze/thawing your primary source. Prepare 10-20 mM working stocks in neutral pH buffer and prepare aliquots that allow you to freeze/thaw the working stock 3-5 times at the most.
Continual freeze/thawing of the primers can cause some break down and this will lead to drift in results such as worsening PCR efficiency and sensitivity. Preparing aliquots will also help avoid contamination problems.
If you accidentally contaminate one of the tubes of primer, you can throw it away and take a fresh one without worrying about contaminating the main stock.
4. Use pipettors accurately for low volumes when making dilutions and adding template
If you require absolute accuracy in quantification and want spot on standard curves, use a pipettor calibrated for low volume pipetting (such as a P2 or P10).
This will ensure reproducibility between replicates and make sure that when you are measuring efficiency based on the standard curve, that you are truly measuring efficiency of the reaction and not your pipetting skill. To learn more about pipetting accuracy, take a look at this article.
5. Perform a standard curve for every new primer pair to check efficiency first
Don’t assume that every set of primers ordered is going to work as well as the last. PCR efficiency can be impacted by a number of factors. A list of critical factors impacting efficiency are listed here.
The best practice is to run a 5 point standard curve with 10 fold dilutions for every new primer pair and make sure you can get at least 90% PCR efficiency with control DNA.
One of the biggest causes of contamination is from using the same pipettors for extraction or handling PCR products post-run for reaction set up. Even if aerosol resistant tips are used all the time, this is a big no-no. Buy a complete set of pipettors that are used for PCR set up and nothing else.
7. Follow the three room rule: separate the rooms for extraction, reaction set up, and cycler location.
In addition to new pipettors, you will want to keep them in a different location, and preferably a different room than the room used for extractions. The ideal set up is to have three rooms; one for RNA or DNA extractions, one for reaction set up (and using a hood with a UV lamp to pre-treat the pipettors and plastics between users), and one for the real-time cycler.
This is the most assured way to make sure you never have amplification in your negative controls.
8. Double check the cycling conditions are correct before starting the run
This is important if you are using a shared instrument. Even if you have your own template file set up, before hitting start, make sure the machine has the correct run cycle for your experiment. Someone may have used your template and made changes to the annealing temperature or the hot start activation time without your knowledge.
Some instruments default back to standard settings and if you are using an instrument for the first time, you may find that your settings didn’t save. It never hurts to double check the run settings.
9. Dilute the template (less is more)
Depending on the gene of interest, you might actually be starting with too much template. Real-time PCR is sensitive enough that sometimes less template gives a more accurate measurement.
You will want samples to cross the threshold between cycles 20-30. Samples that cross the threshold below cycle 15 will fall into most instruments default baseline setting and this will cause a subtraction of fluorescence from the rest of the data.
This can be remedied by adjusting the baseline setting, but if you are unfamiliar with your instrument, it may require a call to technical service to figure it out. Also, if there were any inhibitors in the sample from the purification step (guanidine salts or ethanol, for example) diluting the sample will eliminate their impact on the results and give you an accurate quantitation of the sample.
The best approach for a new sample is to perform a standard curve- even just a 3 point dilution series- to see what concentration will give you a Ct that falls in your standard curve and is most accurate.
10. Make dilutions fresh – do not store dilute solutions unless using a carrier or low retention plastic tubes
Nucleic acids stick to plastic so if you make a dilution series and want to store it for future runs, you will need to protect the samples from absorbing to the tubes walls and becoming diluted out over time.
This can be done by using a carrier nucleic acid, such as tRNA, or by using specially treated plasticware that does not bind nucleic acids. Several manufacturers offer low retention tubes (Axygen is one) or silicon treated tubes to help prevent this occurrence.
If you do store dilutions in non-treated tubes, you may want to re-quant the most concentrated dilutions on a Nanodrop before using to make sure they still match the expected concentration.
Some of these tips may seem like common sense- and they are- to people who have been doing this for a long time. But for many people just starting to use this technology, a lot of time and money can be saved with these simple steps that can make a big impact on results.
There are a lot of resources for real-time PCR help as well, including a very active Yahoo List group and the BioTechniques® Molecular Biology Forums. Fortunately, there are many experts who enjoy helping others master the art of real-time PCR.
And if any experts out there reading this want to list some common mistakes you see in your labs or best practices tips, please let us know in the comments field.
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