2. Any real-time PCR machine is acceptable. Currently, we are using a GeneAmp 5700 thermal cycler (Applied Biosystems) and an iCycler iQ real-time PCR detection system (Bio-Rad).
3. A repetition pipette to distribute the master mix in the reaction tubes is recommended,especially if dealing with many samples, to increase reproducibility and to reduce hands-on time.
4. If you are new to real-time PCR, order a pair of primers (e.g., for a reference gene) that have been shown to work and convince yourself that you can perform the PCR and/or obtain good standard curves.
5. If the exon you are interested in is too small or if no suitable primer pairs can be designed, you can add adjacent intron sequence and develop primers spanning an intron–exon boundary.
6. Some researchers advise sequencing the amplicon; however, this is not always straightforward, because of the small size of the fragment.
7. Sometimes a specific PCR product can generate a (atypical) melting curve with two or more peaks caused by regions in the amplicon that are characterized by different melting temperatures (for instance, a sequence with a GC-poor and GC-rich region).
8. Both annealing and extension can be performed at the same temperature (60°C); there is no need for a separate extension at 72°C during 1 min. Although the extension rate of the enzyme is slower at 60°C, it will certainly be sufficient to extend a 250-bp amplicon at 60°C for 1 min.
9. Having performed 25-μL reactions for many years, we now routinely use a 15-μL reaction. All components of the reaction mix are downscaled in the same proportion,but the amount of template DNA remains unchanged. In this way, we obtain slight increase in sensitivity (lower Ct values, because of the higher initial template concentration) and a 40% decrease in reagent cost.
10. For the PCR master mix, make an excess of one reaction (for <20 reactions) or an excess of 5% (when dealing with >20 reactions). Always run duplicate reactions for each sample, including the no-template control. Work with filter tips in a dedicated PCR workstation (no flow) equipped with UV decontamination bulbs.
11. Prepare the reaction mix (reagents for quantification and primers) in a pre-PCR room to avoid carry-over contamination; this is a room different from the lab in which the DNA is prepared or in which post-PCR manipulations are performed. It might also help to use uracyl-N-glycosylase and dUTP nucleotides in the PCR.
During an initial step at 50°C, the uracyl-N-glycosylase enzyme cleaves contaminating PCR products (carry-over from previous runs).
12. After preparing a 96-well plate for qPCR analysis (reaction mix and added DNA samples), shake the plate on a plate shaker (or vortex) to mix the DNA with the reaction mix, and centrifuge the plate shortly to spin down the reaction mixture and remove air bubbles. Always check the wells for air bubbles, because air bubbles can cause unusual reaction plots and, hence, inaccurate quantification of the gene copy number.
13. In the comparative Ct method, the value of 2 is used as the base in the formula 2ΔCt. A value of 2 means that the reaction efficiency of the PCR was 100%, which is almost never the case. The base value should be adjusted to the actual PCR efficiency (e.g., a value of 1.90 should be used if the efficiency is 90%). Most people determine the reaction efficiency once using a standard curve, and, later on, use
this value in their comparative Ct analytical procedure.
14. Although it is possible to collect fluorescence data at a higher temperature (between the Tm of the nonspecific/primer dimer signal and the true signal; thus,removing the contribution of nonspecific signal to the measurement), this is not recommended because the simultaneous amplification of the nonspecific product(s) can adversely affect the amplification of your sequence of interest.
http://medgen.ugent.be/CMGG/thesissen/Jasmien Hoebeeck.pdf
