In the present study, we evaluated the RT-qPCR assay used to measure the siRNA knockdown of EGFR expression. In our experiments, we were interested in determining the ability of a series of EGFR siRNAs to knock down the levels of endogenous EGFR mRNA expression in a human lung cancer cell line expressing wild type EGFR. To exclude effects of transfection efficiency on siRNA efficacy, we determined H358 cells transfection efficiency with two approaches: cell fluorescence by siGLO Transfection Indicators and cell death induced by TOX Transfection Control. The transfection efficiency was higher than 81% at 48 hrs and 92% at 72 hrs, as accessed by either CellTiter-Blue® Cell Viability Assay, or fluorescence, as assessed by fluorescence microscopy (data not shown). These data suggested that the transfection efficiency was nearly optimal with all siRNAs tested. Of the eight siRNAs examined, s604, s752 and s1247 siRNAs showed the highest efficiency in the knockdown of the EGFR mRNA (Figure ). With primer sets q1 and q3 initially used, the reduction of EGFR transcript levels was not greater than 57% at 48 hrs. We then redesigned a primer set q2 to overlap or encompass the EGFR target site of siRNA s1247. Upon reexamination with primer set q2, which encompasses the target site and using siRNA s1247, a 71% decrease of the EGFR mRNA level was observed. It was the strongest effect observed for all eight siRNAs tested. In contrast, the primer set q2 underestimated the knockdown efficacy in the siRNA s604 and s752 experiments in comparison to what was measured with primer set q1 for the same siRNAs. This primer set q1 amplifies a sequence that locates nearer to the target sequence of s604 and s752. Similar results were observed at 72 hrs: with primer set q2, a knockdown of 72% was found in the s1247 experiments, whereas with the same siRNA q1 detected a 53% knockdown and q3 a 53% knockdown (Figure ).
Figure 1 Determination of EGFR siRNA knockdown efficiency in H358 cells with RT-qPCR. (A) Schematic diagram of the EGFR gene exon (boxes) structure and location of siRNAs and RT-qPCR primer sets. For the siRNAs sequences, see Table 1. For the primers sequences, (more ...)
RT-qPCR amplification of a specific targeted mRNA sequence reflects the integrity of that fragment, but does not necessarily represent an intact mRNA. Our data suggest that the EGFR mRNA fragments amplified by the q1 or q3 primer sets after transfection of s1247, remain more intact despite RNA cleavage and thus result in an overestimation of the amount of remaining intact target mRNA. The relatively higher knockdown read-out with primer set q1, after transfection with s604 and s752, compared to q3, is also consistent with this observation (Figure ). These results thus suggest that s1247 is the most effective of the siRNAs tested.
To further corroborate this hypothesis we analyzed EGFR protein levels with western blot and studied possible phenotypic consequences of EGFR down-regulation in H358 cells. Cell viability and caspase-3/7 activity were measured and, in addition, we evaluated the induction of apoptosis. The aggregate results of these experiments are consistent with the mRNA knock down results obtained in the RT-qPCR experiments and confirm that of the siRNAs tested, the s1247 is the most powerful siRNA on H358 cells to down-regulate the EGFR protein level, inhibit the cell viability and induce apoptosis in comparison to other siRNAs (Table , Figure ).
Phenotypic consequence of EGFR downregulation by siRNAs
Figure 2 Down-regulation of EGFR protein levels and phenotypic consequences of EGFR knockdown. (A) Western blot was performed to determine the down-regulation levels of EGFR protein. (B) Cell viability was detected by CellTiter-Blue® Cell Viability Assay. (more ...)
To verify whether the effect of primer set choice on RT-qPCR results is also observed with other messengers, we transfected a positive, validated GADPH siRNA (Invitrogen, proprietary sequence) into H358 cells and measured the GAPDH mRNA knockdown level with three different primer sets located in different positions. With different locations, the primers detected different knockdown efficiencies (data not shown), and the best primer set was GAPDH 820F and 1106R (Table ). The results from GAPDH RNAi are analogous to the data obtained with the EGFR mRNA suggesting that these findings can be generalized, i.e., measurements of the knockdown efficiency can be influenced by the primer set used for RT-qPCR.
Primer Sequences for EGFR and GAPDH Transcripts used for Real-Time Quantitative reverse Transcriptase Polymerase Chain Reaction
SiRNA induced cleavage of mRNA appears to be followed by degradation of the messenger, which is assumed to be complete 24-48 hrs after siRNA treatment [4
]. Consequently, the position of the PCR primers within the mRNA (cDNA) is generally considered irrelevant, and many publications do not provide a rationale for the primers chosen and specifically do not include qPCR primers that overlap with the siRNA target sequence. However, there are indications that mRNAs are incompletely degraded 24 hrs after transfection as evidenced by others in a Northern blot analysis following treatment with siRNAs against coagulation factor III [2
]. Our results corroborate and strengthen observations made by Shepard et al. [3
]. In a study targeting the connective tissue growth factor in human trabecular meshwork cells, they found that the location of the qPCR primers relative to the siRNA target sequence profoundly influenced the RT-qPCR results, by as much as 60%, 24 hrs post-transfection. They also showed that these results were independent of siRNA concentration, siRNA:lipid ratio, or transfection efficiency. Primer sets that did not overlap the siRNA target sequence yielded a lower knockdown efficiency, which can be explained by the existence of partially degraded mRNA molecules that are still detected in the RT-qPCR assay. However these results favoring a primer position effect were based upon analysis 24 hrs post treatment, a time point at which abundant mRNA may have been cleaved by the argonaute protein, but not yet degraded by exonucleases. More recently, the importance of primer position for assessment of MYCN oncogene siRNA silencing in a human neuroblastoma cell line (IMR-32) was also described by the group of Vandesompele [5
]. Their RT-qPCR analysis was at 48 h post transfection. Our present results strongly suggest that amplification of incompletely degraded mRNA molecules, as observed with the EGFR q1 and q3 primer sets with siRNA s1247, may lead to underestimation of the siRNA efficacy, at least up to 72 hrs post-treatment. Nevertheless, the fact that these position effects were measureable in different cell types (trabecular cells, neuroblastoma and lung cancer cell lines), and with different genes (connective tissue growth factor, MYCN oncogene, EGFR, GAPDH), strongly suggests that incomplete degradation of mRNA following siRNA treatment is a general phenomenon that may significantly affect RT-qPCR results in RNAi research.