RNAi Synthetics Support—Troubleshooting

Depending on the gene you are working with, it can be measured at the mRNA level as soon as a few hours after transfection. We recommend assessing the mRNA knockdown 48 hours posttransfection. Factors affect the timing include the transcription activity, the turnover rate for the mRNA, and if there are alternative pathways. To determine the peak knockdown, it is best to perform a time course experiment.

Please see the following possibilities and suggestions:

• Were the mRNA levels checked? The most reliable method is real-time PCR. In some cases, knockdown of a protein can be affected by other variables, such as protein turnover rate, even though the RNA is knocked down.
• How is the RNA being isolated? Has the quality of the isolated RNA been checked? Ensure that the RNA has not been degraded.
• Was a positive control used? This can help to determine whether the reagents are working and whether the siRNA was delivered correctly to the cell. Run your experiment in parallel with the positive control siRNA.
• Was a transfection control used? What is the percentage of transfected cells? 
• Was a time course used? Generally, gene silencing can be assessed as early as 24 hours posttransfection. However, the duration and level of knockdown are dependent on cell type and concentration of siRNA.
• Was optimization of transfection conditions performed? You can try using different cell densities and siRNA concentrations.
• Which concentration of siRNA did you use? We recommend testing multiple concentrations between 5 nM and 100 nM. 

Please see the following possibilities and suggestions:

• How many siRNA did you test? Is there any knockdown? If there is no knockdown (<10%) in any of the siRNA, then the assay is likely the problem. Try using a different qRT-PCR assay to assess knockdown.
• What was the positioning of the qRT-PCR assay target site relative to the cut site for the siRNA? If greater than 3,000 bases away, the problem could be alternative splice transcripts.
• What are the C_ts for the experiment? They should be below 35 in a 40-cycle qRT-PCR experiment.
• Did you confirm the siRNA got into the cell? We recommend using a validated positive control siRNA to check the transfection efficiency. 

In some cases, knockdown of a protein can be affected by other variables such as protein turnover rate, even though the RNA is knocked down. Additionally, a longer time course may be needed to see an effect on protein compared to mRNA.

Please see the following:

(a) Silencer® Select: “Life Technologies guarantees that when you purchase two Silencer® Select Pre-designed siRNA to the same target, then those two siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at ≥5 nM and mRNA levels detected 48 hours posttransfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer® Select siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product.”

(b) Stealth RNAi®: “Life Technologies guarantees that when you purchase three Stealth RNAi® Pre-designed siRNA to the same target, then at least two of those three independent, non-overlapping siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at ≥20 nM and mRNA levels detected 48 hours posttransfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Stealth RNAi® sequence will be synthesized free of charge. This guarantee does not extend to any replacement product. We also recommend the use of an appropriate negative control, such as one of the three Stealth RNAi® Negative Controls to normalize message knockdown.”

(c) Silencer®: “Life Technologies guarantees that when you purchase three Silencer® Pre-designed siRNAs to the same target, at least two of the siRNAs will reduce target mRNA levels in cultured cells by 70% or more when measured 48 hours after transfection at 100 nM or higher final siRNA concentration under the conditions described below. If at least two of the three siRNAs do not induce >70% target mRNA knockdown, Life Technologies will provide a one-time replacement of up to three Silencer® Pre−designed siRNAs per target at no additional charge. Requests for replacement product must be made within one hundred and eighty (180) days from the date of delivery of the Silencer® Pre-designed siRNAs. Optimum transfection efficiency must be confirmed using good laboratory practices and a proven-to-work siRNA to an endogenous message, such as the Ambion® Silencer® GAPDH siRNA Control. To assess knockdown, target mRNA levels in treated samples must be compared to that of cells transfected with a nontargeting control siRNA, such as Silencer® Negative Control #1. We recommend TaqMan® Gene Expression Assays to quantify mRNA levels.

All Validated siRNA are guaranteed individually to knock down the target mRNA by either 80% (Silencer®) or 70% (Stealth RNAi®).