TechNotes 8(3) The Ambion Top Ten

The 10 Most Common Quantitative RT-PCR Pitfalls

1. Quantitation performed when products are not in exponential phase. For relative RT-PCR data to be meaningful, the PCR must be terminated at a cycle within exponential phase for all products of the reaction (before reagents become limiting, and before product renaturation starts to compete with primers for annealing).
   
   
2. Not using a "minus RT" control in each experiment. The minus RT control indicates whether DNA is contaminating the RT-PCR reaction. The presence of even trace amounts of DNA can generate false positive products amplified during the PCR step.
   
   
3. Using an inappropriate internal control. The expression level of a good internal control should not vary across the samples being analyzed. However, the expression level of many commonly used internal controls, such as §-actin and GAPDH, frequently does vary across tissue type or experimental conditions. Ambion recommends using 18S rRNA as an internal control because its expression level is invariant over a wider range of experimental conditions than other traditional internal controls. See Technical Bulletin #151 for a detailed discussion on this topic.
   
   
4. Using EtBr to quantitate results. EtBr staining is a poor method to quantitate relative RT-PCR; it has a dynamic range of only 1/2 to 1 1/2 logs meaning you can only detect a 50 fold difference in expression across samples. Switching to more sensitive staining agents or labeling with ³²P greatly increases the dynamic range and thus the expression difference you can detect.
   
   
5. Not using "intelligent" primer design. Primer design software will help design robust primers that avoid regions of high secondary structure. It is also advantageous to choose primers that span an intron:exon boundary to prevent amplification from contaminating DNA.
   
   
6. Introducing DNA cross-contamination. All surfaces in the PCR work area should be routinely decontaminated to prevent cross contamination.
   
   
7. Not setting the base-line properly in real-time PCR. To obtain accurate C_T values, the baseline needs to be set two cycles earlier than the C_T value of the most abundant sample.
   
   
8. Using a DNA mimic for competitive RT-PCR. DNA mimics (which use sequences unrelated to the target) fail to control for differences in RT efficiency and amplification efficiency. The ideal exogenous standard is an RNA molecule that is identical in sequence to the endogenous target except for a 10% internal deletion. See Technical Bulletin #185 for more information on this subject.
   
   
9. Using poor quality RNA. Degraded or impure RNA can limit the efficiency of the RT reaction and reduce yield.
   
   
10. Not using "cocktails". A "cocktail" (a mixture of the reaction reagents) should be used when setting up multiple reactions to minimize sample-to-sample variation and improve reproducibility.

10 Products for Quantitative RT-PCR

1. QuantumRNA™ 18S rRNA Internal Standards. The ideal internal control for quantitative RT-PCR.
   
   
2. Gene Specific Relative RT-PCR Kits. Gene specific primers for human, mouse, and rat genes paired with QuantumRNA 18S rRNA Internal Standards.
   
   
3. DNA-free™. DNase Treatment & Removal Reagents easily eliminate DNA contamination, and later DNase, from RNA samples.
   
   
4. DNA Molecular Weight Size Standards. For accurate sizing of PCR products.
   
   
5. DNAZap™ PCR DNA Decontamination Solution. Instantly removes DNA and RNA contamination from surfaces.
   
   
6. PCR Tubes. Thin-walled PCR tubes guaranteed RNase- and DNase-free.
   
   
7. RNAqueous™-4PCR. DNA-free RNA isolation kit for rapid, phenol-free total RNA isolation.
   
   
8. FirstChoice™ RNAs. High quality DNase-treated human, mouse, and rat total and poly(A) RNAs optimally suited for RT-PCR.