Download these files:
Excel Template for real time PCR
News:
10/5/2005 - New TaqMan style PCR details have been added to the worksheet. It's so easy that we are switching over to this method for routine PCR work.
10/2/2005 - Many have been asking questions about RNA or DNA contamination. I added a whole new bookmark/chapter for this discussion alone.
10/2/2005 - TaqMan style PCR method added. Now you can design dual-labeled probes to match those from the literature and run a TaqMan-style PCR on the cheap!
9/30/2005 - Please participate in the message board. Ask your questions there instead of emailing me. Thanks!
9/30/2005 - Added a formula for manual calculation of the standard curve. This is important if you have more than 96 samples.
9/28/2005 - Fixed a bug in the excel template that affected your master mix calculation if you changed the RNA quantity. Please download the new version.
8/31/2005 - New message board added. If you've designed new primers, post them here to share with everyone. I'll also post questions and responses here.
8/30/2005 -
MAJOR revisions to the real time worksheet. Now it includes ordering information (product name, catalog number, price, and even company info) for all the products I recommend. I added a FAQ to the worksheet.
8/24/2005 - I have updated the excel template. Please download the new file. It now contains a new section for Superscript III (Invitrogen). This reverse transcriptase is great for low-quantity samples. I use it for all animal tissues now. I still use MMLV-RT from Promega for doing cell culture samples, with great results.
I'm currently working on making artificial TaqMan style probes. Once I work out the conditions, I will post the instructions. If anybody has tried this, please let me know your results. It should save a ton of money by allowing us to buy a generic PCR master mix, instead of the SYBR green mix, which is quite expensive.
This webpage is now #27 in the yahoo search engine for real time PCR!!
Also, the PCR primer list is seriously out of date. I will try to fix that in the next week.
There is a great review article in Biotechniques about real time PCR (link). I will post the link later...
About the author
I am a post-doctoral fellow working in the lab of Jeffrey Laskin at Rutgers University. We investigate the role of oxidative stress generated by a variety of toxicants in mediating their toxicity. Please see my webpage for more information and relevant abstracts. Or, email me.
Regular PCR versus Real Time PCR
PCR-based methods for the detection of changes in gene expression are
powerful techniques. Traditional quantitative RT-PCR analyzes
differences in gene expression by comparing samples with equal starting
amounts of RNA. After optimizing for the number of cycles (and
other parameters), the products are separated on a gel, and compared
with an internal control. [insert Jack's link here]. While
this method is good, products are seen after the exponential phase of
amplification, which lowers the sensitivity. For more information
on the basics of PCR, see [this link].
Real-time PCR is an improvement on regular PCR.
1. Products are measured after each cycle, rather
than at the end of the run. This eliminates the need for
determining the optimal cycle number (all PCRs are run for 40
cycles).
2. A standard annealing temperature is used,
eliminating the need for optimization. Nearly all primers are
designed to work at this temperature.
3. Depending on the dye used, the sensitivity is
increased by several orders of magnitude.
4. Samples are analyzed during the exponential phase,
where differences in quantity of products are maximized.
This web page will describe a standard method (SYBR Green and TaqMan) for real-time PCR, starting with cell culture or animal tissue. Sample preparation, reverse transcription, primer design, materials needed, and other methods will be covered. Analysis of data will also be described. Please email me if you find any errors in this webpage.
This guide is a basic guide. There are many nuances to doing PCR that will not be covered here. The information on this web page is for informational purposes only. Use at your own risk. The author(s) assume NO RESPONSIBILITY for any errors on this page.
The Applied Biosystems 7900HT high throughput real time PCR machine.
SYBR Green Versus TaqMan
The principal advantage of the SYBR green method is cost. The TaqMan method is superior in terms of data, time required for quality control, and performance. If you are investigating many different genes, I would suggest the SYBR green method.
The SYBR green technique uses a dye that binds to any double-stranded piece of DNA. Similar to ethidium bromide, this dye will increase in fluorescence as product accumulates in the reaction. However, SYBR green cannot differentiate between products. Therefore, primer dimers and spurious products will also be detected. Because of this, a number of controls are required for SYBR green determination. These methods will be described in this website.
The TaqMan method is superior. This technique uses three primers instead of two. Two primers amplify the product as usual. The third primer is a dual-labeled probe containing a fluorescent emitter and quencher. As product accumulates in the tube, the third primer will bind to that product. The 5' exonuclease activity of Taq destroys this third primer, separating the fluorescent emitter and quencher, resulting in increased signal. Because the third primer will only bind to product, it is irrelevant whether other products are forming in the reaction.
A third method is sold by Invitrogen. This method uses a LUX probe attached to the forward primer. A hairpin loop in the primer quenches the dye. When incorporated into a product, the quenching is removed, increasing fluorescence. I have not tested this method, although it is cheaper than TaqMan, and more expensive than SYBR green.
There are new methods out there as well, but SYBR green and TaqMan are the easiest to start with.
All PCRs are run on Applied Biosystems real-time PCR machines. A publicly available real-time PCR machine is located in UMDNJ's sequencing facility. All PCRs that I have run so far have used Applied Biosystems reagents. Please email me if you have a protocol or experience with other companies' reagents. [Materials]
I have compared SYBR green master mix ingredients from Takara, Applied Biosystems, and Qiagen. Here are the results (coming soon):
RNA and cDNA Sample Preparation
RNA can be prepared using a number of different methods. It is extremely important to have good quality RNA. The following methods describe RNA preparation from various sources. I highly recommend Qiagen for RNA preparation.
cDNA Generation
Rather than do reverse transcription PCR (RT-PCR), my protocols generate the cDNA ahead of time. The cDNA generation protocol is the standard one from Promega's MLV-RT. See this guide. cDNA Generation
The real-time calculator (on the left bar) - this is a spreadsheet that lets you plug in the number of samples, and gives you the required components. It gives reaction conditions for cDNA generation, and for the SYBR green real time PCR reaction. It also gives you product information for ordering.
RNA or genomic DNA contamination of your samples - NOT AN ISSUE
There are two options: degrade them or ignore them. I offer arguments for both below:
Degrade them: Some genomic DNA is carried over in the preparation of your RNA and cDNA. One way to avoid issues with this is to design primers that overlap intron/exon splicing sites. This way, there is no chance that any of the amplified product you see is an artifact of genomic DNA. Qiagen sells an on-column DNase for use in their RNA preparation kits, although it is expensive.
Carryover RNA contamination is more of a problem. RNA can be amplified by Taq DNA polymerase. One solution is to treat your cDNA samples with RNase, and then inactivate this enzyme in a subsequent step.
Ignore them: This may seem to be inappropriate, but let me present my argument, and then you can decide for yourself.
I don't believe that elmination of RNA or genomic DNA is necessary for the following reasons:
1. You have basically the same amount of genomic DNA and RNA contamination throughout your samples.
2. Both are generally small relative to the concentration of your cDNA.
3. Genomic DNA will be cancelled out because it is equally present in your treatment versus your control sample. This is, however, an issue if you are doing true concentration determinations using a real standard curve with known numbers of molecules of target.
4. Contaminating RNA is present in the same ratio as the cDNA you prepared. I mean, if the RNA was induced 10-fold in your target sample versus your control sample, that ratio is present in both the RNA and cDNA. Therefore if your polymerase amplifies this RNA, so what? The ratio is what you are after anyway!
5. Finally, if they are present, they will only make your data look worse, not better. Remember, in your reaction, you loaded equal concentrations of RNA to your cDNA generation reactions. You have already controlled the amount of starting RNA present.
6. The best proof is your positive control genes. For example, if COX-2 is induced by LPS in your sample, like it's suppossed to be, then you know your PCR has worked well, and that your samples are OK. This is why a positive control treatment and target gene are the MOST IMPORTANT things on your PCR.
The best tool for designing primers is the Primer Express Software, which is sold by Applied Biosystems. Nearly 90% of the primers I have designed using this software are acceptable. A copy of this software is installed in C.S. Yang's laboratory on the second floor of the Cancer Institute.
In my experience, other primer design programs, which are used for typical PCR, are not acceptable. Alternatively, you can search the literature for published primers. About 50% of the primers I use from the literature were successful. Some primers are published on a public database [here].
Another alternative is to design TaqMan primers. These are designed using a free program on Applied Biosystems' website. Although more expensive than the SYBR Green method, their primers are guaranteed to work (or they will give you another pair to try).
Basic technique to design SYBR green primers using Primer Express
Primers can be ordered through IDT, through UMDNJ's sequencing facility (www.idtdna.com). Typically, I get 25nmole scale, just desalting, and they run about $5 each. For oligo dT, order 1umole, standard desalting, for about $22.
To order TaqMan style primers, find your sequences in publications, or design them using the Primer Express software. You will be ordering three separate DNA sequences: two primers, and one internal probe. The internal probe is a dual-labeled probe. You can either order this through Applied Biosystems, or order a dual-labeled probe from a company, such as IDT. Order a 5'-FAM and 3'-Black Hole Quencher to match a TaqMan style approach.
Testing your Primers - Your First PCRs
Before running an actual PCR with your samples, you must test your primers and samples to make sure they are adequate. They are:
1. Test your primers using a standard curve generated from your samples, and a water blank.
2. Run a dissociation curve to indirectly test for multiple products.
3. Run a RNA sample to test for genomic DNA contamination.
4. Run some product on a gel and make sure your product(s) are the correct size. (some labs don't do this).
5. Subclone your product into a T vector (such as the pGem T-Easy kit from Promega), and sequence ten products to make sure that the product is correct. This is the only way you can be absolutely sure that you are amplifying the correct product, and that a second product isn't the same size as your presumed target. (most labs don't do this).
First, run a test PCR with your primers. You can do this with a few of your samples, and by running a standard curve. A standard curve that works shows you that both your samples and your primers are good. Here is an exact protocol for setting up your standard curve. [protocol].
When loading your plate into the real time PCR machine for the first time, have someone help you set up the machine. The program needs to be told which dye it is reading. If you don't select one, none is read. You then lose your data. Also, be sure to add the dissociation protocol to the program. This will help show whether your primers are good.
This is a guide that will help you through the process of analyzing your results. It will help you determine whether your run has been completed successfully. [guide]
Housekeeping genes are often used to control your data against to check for equal loading and reverse transcription. When choosing your housekeeping gene, you want a gene that does not change with your treatment. There are many examples throughout the literature of housekeeping genes. The three I will focus on are beta-actin, GAPDH, and 18S rRNA. A good list of housekeeping genes can be found at Invitrogen's website. This page will talk about the advantages and disadvantages of these three housekeeping genes.
Facilities at Rutgers and UMDNJ for performing real-time PCR:
Andrew Brooks, Ph.D., is the new Director of the Bionomics Research and Technology Center at RWJMS and Rutgers University. His facility specializes in microarray and real-time PCR applications on a large scale. His website will soon be available at: http://EOHSI-BRTC.com
Andrew Brooks, Ph.D
Director, Bionomics Research and Technology Center
Robert Wood Johnson Medical School
Bionomics Research & Technology
Environmental and Occupational Health Sciences Institute
Suggeted Reading
See this article in Biotechniques: Real-time PCR for mRNA
quantitation
Marisa L. Wong and Juan F. Medrano
BioTechniques Vol. 39, No. 1: pp 75-85 (July 2005). Free with
registration.
Pitfalls of QRTRT-PCR. Bustin et. al. http://jbt.abrf.org/cgi/content/full/15/3/155
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12184808
RNA preparation
http://www.abrf.org/Other/ABRFMeetings/ABRF2002/RNAprimer.pdf
Great forum for discussing real time PCR and other related issues:
http://micro.nwfsc.noaa.gov/forums/
