Analysis of multiple targets on the same plate requires that all primer sets work using the same thermal cyclingparameters. The primer pairs for the cytokine targets were selected using the PE Biosystems Guidelines for‘Amplifying Custom Target Sequences for Quantitation’.15 Following these guidelines allows the amplification ofall targets to be performed using the same reaction conditions and thermal cycling parameters. Adhering to theseguidelines also means that the efficiency of each PCR amplification is close to 1. Because the efficiencies of thedifferent amplifications are approximately equal and close to 1, it is possible to obtain relative quantitative resultswithout having to run standard curves for any of the cytokine targets. Detailed procedures for relative quantitationare described in ABI PRISM® 7700 Sequence Detection System User Bulletin No. 2.16 Figure 5 shows the relativequantitation results comparing the four samples for the three cytokine targets. In this analysis, the untreatedsample was designated the calibrator and it is assigned the value of 1´ for each of the targets. The quantity of eachcytokine mRNA in each of the treated samples is given relative to the calibrator sample.
Figure 5. Relative quantitation results for three cytokine mRNAs in PBMCs that have been stimulated with CD3/CD28 antibodies. (Data generated in collaboration with DNAX Research Institute.)
The results shown in Figure 5 were normalized using the CT values obtained for the ribosomal RNA amplificationsrun in the same plate. The 18S ribosomal RNA is an endogenous control that is used to normalize the samplesfor differences in the amount of total RNA added to each cDNA reaction. The use of this endogenous reference alsonormalizes for variation in reverse transcriptase (RT) efficiency among the different cDNA reactions. Variation inRT efficiency other than sample-to-sample variation is controlled for because a single cDNA reaction is performedfor each total RNA sample. This single cDNA reaction is then split to perform the target and control amplifications.
At high levels of RNA, Figure 5 shows that approximately the same relative quantitation results are obtainedwhether SYBR® Green I dye detection or fluorogenic probe detection is used on either the 5700 or 7700 system.Thus, at the 2 hr time point, the increase in cytokine mRNA level is approximately 2000-fold for IL-2, 100-fold forIL-4, and 40-fold for TNF-a, regardless of analysis method used. At low levels of RNA, though, the detection ofnon-specific amplification by SYBR® Green I dye complicates the results. For the IL-2 analysis in the overnightsample, fluorogenic probe detection on either the 5700 or 7700 system shows the level of IL-2 mRNA is about0.75´ the amount in the untreated sample. Using SYBR® Green I dye detection, the amount of IL-2 mRNA in theovernight sample appears to be 3.5´. This inflated value observed with SYBR® Green I dye detection is due to thedetection of non-specific amplification products. The TNF-a levels observed in the overnight sample reveal adifferent consequence of non-specific amplification. Both analyses using fluorogenic probe detection indicate theTNF-a level is about 0.6´ compared to the untreated sample. The two SYBR® Green I dye results give wildlydifferent values of 1´ and 0.03´. Non-specific amplification is not necessarily consistent well-to-well, so replicatescan give much different results. Thus, non-specific amplification can lead to erroneous and/or highly variable resultsat low target levels when SYBR® Green I dye or another generic DNA-binding dye is used for detection. For theseparticular cases, lower primer concentrations were tried to increase the specificity of the amplifications. Changingprimer concentrations from 900 nM to 50 nM reduced the spurious results observed with SYBR® Green I dye detection.
