PCR-CUCOs: Customised Competitors for Quantitative PCR and RT-PCR 

For the quantification of nucleic acids methods of competitive PCR using heterologous sequences for calibration are well established [1-4]. Now you can order customized heterologous competitive internal standards for quantitative PCR (Competitor DNA) [5] and quantitative RT-PCR (Competitor RNA) [6]. Serial dilutions of known quantities of competitor DNA (or RNA) are added to PCR- (or RT) reactions containing constant amounts of sample target. As both the competitor and your specific target are amplified by the same primers, they compete during the amplification reaction. PCR products are resolved by gel electrophoresis and the quantities of competitor and target PCR product are compared. The target amount can be determined from the competitor dilution in which the amount of competitor and target PCR product are equal. We recommend to select a competitor which is at least about 10% larger or smaller than your target PCR fragment. Various sizes of competitor molecules can be ordered to take care of your special requirements.

You provide us with the sequence of the forward and the reverse primer for your quantitative (RT)-PCR experiment, and Mobidab manufactures your specific competitor of choice.

Example:

Quantification of rat muscle fructose-1,6-bisphosphatase mRNA by competitive RT-PCR.

Quantitative RT-PCR was performed using 20 ng of total RNA from rat muscle which were titrated with four different known amounts of the heterologous competitor RNA (2 x 10e4, 4 x 10e4, 6 x 10e4, 8 x 10e4 molecules, lanes 1 to 4), reverse transcribed and amplified. Competitor RNA was obtained by in vitro transcription from complete competitor DNA (Comp400 containing the binding sites for forward and reverse primer) usingT7 RNA polymerase.

Figure 1: Results of agarose gel electrophoresis. 10 µl of the PCR products were applied per lane. The gel was stained with ethidium bromide.

Figure 2: Plot of experimental data and resulting linear regression curve. The logarithm of the ratio of the volume of fluorescence intensity per base pair (FI) of the competitor and target RT-PCR products are plotted against the logarithm of the number of competitor RNA molecules. The number of target RNA molecules is determined from the point of intersection of the regression curve with the X-axis (equivalent fluorescence intensities of RT-PCR products of target and competitor). 1.7 x 10e6 molecules of muscle fructose-1,6-bisphosphatase mRNA are present in 1 µg of total muscle RNA.

We recommend to select a competitor which is at least about 10% larger or smaller than your target PCR fragment. Various sizes of competitor molecules can be ordered to take care of your special requirements.
You provide us with the sequence of the forward and the reverse primer for your quantitative PCR or RT-PCR experiment, and Mobidab manufactures your specific competitor of choice.

You can choose from:

Complete competitor DNA (For quantitative PCR and for generating competitor RNA for quantitative RT-PCR)
Competitor DNA (For quantitative PCR)
Competitor RNA (For quantitative RT PCR)

Figure 3: Structure of complete competitor DNA, competitor DNA, and competitor RNA
Legend:
T7: T7 Promotor
FP, RP: Forward and Reverse Primer

The insert is prokaryotic DNA with 50 % GC content, homogenously distributed.
The DNA or RNA concentration will be determined by UV spectroscopy and will be given in ng/µl on the accompanying quality control data sheet.

We recommend to select a competitor which is at least about 10% larger or smaller than your target PCR fragment.The following sizes are available:

Comp 180: Amplificate Size 180 bp + Primers
Comp 285: Amplificate Size 285 bp + Primers
Comp 400: Amplificate Size 400 bp + Primers
Comp 515: Amplificate Size 515 bp + Primers
Comp 630: Amplificate Size 630 bp + Primers
Comp 745: Amplificate Size 745 bp + Primers
Comp 975: Amplificate Size 975 bp + Primers
Comp 1090: Amplificate Size 1090 bp + Primers

References:

[1] Haberhausen, G, Pinsl, J, Kuhn, C, Markert-Hahn, C Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays. Journal of Clinical Microbiology, 1998 March, p. 628-633, Vol. 36, No. 3

[2] Auboeuf D, Vidal H The use of the reverse transcription-competitive polymerase chain reaction to investigate the in vivo regulation of gene expression in small tissue samples. Anal Biochem 1997 Feb 15;245(2):141-8

[3] Vats A, Katayama H, Kim Y, Mauer M, Fish AJ, McGlennen, RC Modified Method for Competitive Reverse Transcription Polymerase Chain Reaction for Rapid and Automated Quantitation of Messenger RNA in Multiple Samples. Mol Diagn 1997 Dec;2(4):235-240

[4] Gopalakrishnan R, Morse MA, Lu J, Weghorst CM, Sabourin CL, Stoner GD, Murphy SE Expression of cytochrome P450 2A3 in rat esophagus: relevance to N-nitrosobenzylmethylamine. Carcinogenesis 1999 May;20(5):885-91

[5] Siebert PD, Larrick JW. PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR. Biotechniques 1993 Feb;14(2):244-9.

[6] Borriello F, Lederer J Construction of quantitative RT-PCR MIMICs. Biotechniques 1995 Oct;19(4):580-2, 584