Quantitative PCR which meets mathematical ideals

[Peter Doris]

I regret that I have missed some of the early discussion on quantitative 
RT-PCR which seems to have generated much interest.

Raeymaekers has done much to generate realistic mathematical models of 
these reactions. Readers should also note his recent paper in Genome 
Research 5:91-94, 1995.

The real problem is that there has been no description of this method 
which has been extensively validated and shown to meet not only the 
theoretical requirements, but also demonstrated to accurately and 
absolutely quantify RNA and DNA inputs. Raeymaekers' work has been 
thorough and helpful on theoretical grounds and he has done much to 
point up the considerations which must be met, though it has remained 
unclear whether and how such considerations can be met.

This situation will change with a forthcoming publication which is 
principally the work of Amanda Hayward-Lester (BioTechniques, in press). 
This work has used novel HPLC column technology developed by Dr. Peter 
Oefner (BioTechniques 16:898-908, 1994) to rapidly separate and 
quantitate products of competitive RT-PCR reactions. The principal 
findings are that titrations with slopes and R^2's close to unity are 
routinely achievable using this system. HPLC provides some important 
advantages which play a role in permitting quantitative accuracy. First, 
there is no need to label reaction products, they can be detected and 
quantified by UV absorbance as they elute from the column. Second, there 
is no need for post-reaction sample processing prior to HPLC analysis - 
reaction products can be directly injected into HPLC. Third, this 
technology reveals aspects of competitive reaction kinetics which cannot 
be seen when the same reaction products are analyzed on either agarose 
or polyacrylamide gels. This new information is important to provide 
accurate titrations. What is made clear by rversed phase ion-pair HPLC 
is that these competition reactions readily generate heteroduplex 
products, even when the native and competitor sequences share less than 
65% homology. On non-denaturing gels we have not been able to visualize 
these heteroduplicies as separate products (on denaturing gels, they 
become irrelevant to the computation). This appears to be due to the 
peculaiar migration of heteroduplexes which effectively contain both 
double and single stranded DNA. Since the quantitation system depends on 
the ratio of products and the presence of heteroduplices affects this 
ratio, it is vital not only to detect them, but also to quantitate them 
so as to calculate an accurate ratio. The system reveals that if native 
RNA is assayed at dilutions varying over three orders of magnitude, each 
titration produces a line with near ideal slope and R^2 properties and 
each line is displaced in parallel from adjacent lines by the degree 
expected to result from the dilution of input native RNA.

Further work has confirmed the reliability, accuracy and precision of 
this method. For competitor templates in the range of sizes examined so 
far with this system, PCR amplification efficiency of native and 
competitor inputs and the decline in efficiency as the reaction proceeds 
through the exponential and into the saturation phase of the reaction 
are identical - no other conclusion can be supported by the data. In 
addition, the ability to generate absolutely accurate estimates of gene 
expression appears confirmed by experiments in which known quantities of 
both RNA and DNA have been input into titrations and the estimated 
amount of RNA or DNA has been compared to the known initial input. New 
insight has been gained into optimal characteristics of mutant 
competitor templates for use in this system. Most importantly, the 
possibility of quantification without routine, laborious, extensive 
titrations emerges as a probability.

The HPLC column is a reversed-phase, non-porous polymer. It is now 
commercially available from Sarasep, Inc, Santa Clara, CA (408 492 
1029). I have no commercial connection with Sarasep, though I have been 
involved in developing this technology with their knowledge and 
co-operation. I am also a co-author on the forthcoming BioTechniques 
paper.

On a related subject, if you would like to try some competitive PCR 
simulation, I have reconstructed Raeymaekers' model in a spreadsheet. 
Please send me e-mail (hepad at ttu.edu or cbbpad at wpoffice.net.ttuhsc.edu) 
and I will send you a copy of the spreadsheet. It is an Excel 5.0 
binary.