latest news

07/15/09

Welcome to the new version of the real time PCR website. Little information has changed, but the primer list has been updated!

 

getting started

Your first step is to purchase the required equipment, supplies, and reagents.

Real Time PCR Machine

You can't do real time PCR in a standard PCR machine. You need a real time PCR machine that can take optical measurements of your samples after each cycle. I've had direct experience with Applied Biosystems and with Bio Rad, both of which are leaders in the industry. As of right now, you can expect to spend about $30,000 for a machine.

Supplies

To perform real time RT-PCR, you will need a source of RNA, a means of extracting the RNA, a reverse transcription kit, specially designed primers, and reagents to run the actual PCR, The following sections will describe each of these steps.

Source of RNA

Your source of RNA could be tissue or cells grown in culture.

Means of Extracting RNA

There are several options when it comes to extracting the RNA. Trireagent from Sigma is the traditional method and the cheapest. This reagent uses phenol to extract RNA from the DNA and protein. The advantage of this is the cost, and the disadvantages are that it is time consuming and produces dangerous waste. However, for rare or valuable samples that have little RNA, it may be the best.

My favorite mechanism of extraction is the RNeasy kit sold by Qiagen. It costs about $4 per sample to extract using this method, but it is very fast and provides great quality RNA.

A shortened protocol for animal tissue extraction can be found here. Preparing RNA from immortalized cell lines can be found here.

cDNA preparation

The next step is to convert your mRNA into cDNA. I recommend using a kit. A brief protocol is here.

Primers

Primers must be specially designed for real time PCR because the amplicon is much shorter than for traditional PCR. The typical size is approximateley 50 bases. Ideally you should use the program that comes with your real time PCR machine, such as Primer Express offered by Applied Biosystems. If that is not available, there are several commercial websites that offer primer design. One of these is IDT, Integrated DNA Technologies, which provides this for free. Another possibilty of course is to find published primer sequences in a paper and to order those. However, only about 50% of the primers I find publish work for me, so I typically order them myself. Note that IDT's website contains an option to have the primers span intron/exon overlap sites. This is important to use because it prevents amplification of any contaminating genomic DNA in your sample.

Now is a good time to talk about TaqMan versus SYBR green as technologies for measuring your cDNA. SYBR green is cheaper, because it only uses regular primers. Typically these primers can be ordered for $10 per pair. Another alternative is to design TaqMan primers. These are designed or purchased from Applied Biosystems' list of validated primers, and they guarantee them to work. Finally, you could try to find TaqMan primer sequences published in the literature and make your own, using a dual-labeled probe from your primer supplier. For example, IDT sells Fam and Black Hole Quencher as two dyes that you can label your probe with. Of course, there is no guarantee that this will work.

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 for approximately $200.  Order a 5'-FAM and 3'-Black Hole Quencher to match a TaqMan style approach.  

So when is it good to order TaqMan primers?  If cost is a concern (like it is for most academic labs), order TaqMan style probes only for genes that you will measure often.  If you are just exploring to find novel targets, use SYBR green because it is cheaper. 

You can find a list of my own primers here.

Reagents to Run the Reaction

For my PCR runs, I've exclusively used Applied Biosystems SYBR Green Master Mix or Taq Man Master Mix. A few years back I tried Takara's SYBR mix and Qiagen's and saw no difference in SYBR green amplification using the same set of primers.