In multiplex real-time PCR, pairs of primers with different markers are added to a single reaction tube, allowing simultaneous analysis of several different genes. The LUX primers have proved to be very effective in many reactions where the template was quantified by labeling the housekeeping gene with JOE and the target gene with FAM. In the standard multiplex system, the primer concentrations used and volumes added are the same as when performing a mono reaction, as follows. This experiment was obtained from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Multivariate real-time PCR experiment
Materials and Instruments
PCR Super Mix-UDG ROX Dye Distilled Water Reverse Primer Forward Primer Move I. Materials For more product details, please visit Aladdin Scientific website.
ABIPrism 7700 Model Series Assay Systems
1. Buffers, solutions and reagents
Take 50ul reaction system as an example.
Platinum Quantitative PCR Super Mix-UDG (Invitrogen11730-025) 25ul
ROX dye (Invitrogen12223012) 1ul
Sterilized distilled water (Gibco15230-162) 10ul
10umol/L reverse primer I 1ul
10umol/L forward primer I 1ul
10umol/L reverse primer II 1ul
10umol/L forward primer II 1ul
Subtotal 40ul
Template dissolved in 0.1XTE or sterile water 10ul
Total 50ul
2. Specialized instruments
ABIPrism Model 7700 Series Assay System.
II. Methods
1. The temperature conditions during PCR cycling were the same as in Scheme 2
Typical multiplex amplification results are shown in Figure 15-3 (see color figure on cover 2). The experiment used interleukin-4 as the target gene and actin gene (housekeeping gene) as the reference gene. The results showed that when β-actin gene was 106 copies , the quantification paradigm for interleukin-4 was 22~3X15 copies. The PCR conditions were not further optimized, and the concentration of reagents in the system was exactly the same as that in the mono amplification. In case the efficiency of real-time PCR decreases, the PCR system can be optimized according to the following steps.
2. Optimize the conditions for the multiplex reaction
a. For high flavor genes (especially housekeeping genes), the primer concentration can be reduced to 1/4 of the primer concentration of other genes.
b. Increase the MgCl2 concentration from 3 mmol/L to 6 mmol/L.
c. The dNTP concentration for each gene is increased to 400 umol/L.
d. Double the amount of polymerase (0.06U per 1ul in the reaction system). PlatinumTaqDNA Premium Polymerase (Invitrogen10966-018) may also be used.