Skip to main content
Premium Trial:

Request an Annual Quote

Genome Technology s Lab Notebook: Reader Tips and Experiences: Mar 24, 2005

Following is a scientist's responses to the question, "If an RNAi experiment results in insufficient gene knockdown, do you try the experiment again? If so, what approach do you use to improve the knockdown?"

 

For a complete list of scientists' responses, read the March issue of Genome Technology, a GenomeWeb News sister publication.


Typically, insufficient knockdown in our hands results from some of these main factors (alone or in combination):

 

1. Stable protein which has a long half life will be hard to knock down in a timely manner. There is not much you can do about this other than plan the experiment to measure further time points.

 

2. Low transfection efficiency can also lead to overall low knockdown. Optimizing the transfection agent or method, cell growth rate, and other conditions for the transfection can usually help but some cell lines are difficult to transfect, limiting the extent of knockdown.

 

3. Not all siRNA target sequences are ideal, and in many cases, several need to be tested to find one that gives optimal results. In general, the success rate with siRNA designed with the most up-to-date target selection rules will be higher than 50 percent, but this is very gene sequence-dependent and there may be limits to the achievable knockdown in certain genes where there simply are no good target sequences.

 

4. When all else fails, it may be the purity of the siRNA, or its integrity. We favor high-quality synthesis of siRNA from reputable vendors, and we avoid pooling our siRNA to avoid problems associated with off-target effects of bad siRNA in a mix, and to avoid needing to de-convolute a mixture to figure out which siRNA target sequence worked best. The storage of the siRNA and handling may also lead to degradation of the siRNA, and sometimes the simplest solution is to use control siRNA and reorder siRNA that appear to be degraded.

 

If an siRNA is not giving the desired knockdown, one should avoid increasing the concentration too high as this may lead to non-specific effects.

 

Spyro Mousses

Director, Cancer Drug Development Laboratory

Translational Genomics Research Institute

The Scan

Genome Sequences Reveal Range Mutations in Induced Pluripotent Stem Cells

Researchers in Nature Genetics detect somatic mutation variation across iPSCs generated from blood or skin fibroblast cell sources, along with selection for BCOR gene mutations.

Researchers Reprogram Plant Roots With Synthetic Genetic Circuit Strategy

Root gene expression was altered with the help of genetic circuits built around a series of synthetic transcriptional regulators in the Nicotiana benthamiana plant in a Science paper.

Infectious Disease Tracking Study Compares Genome Sequencing Approaches

Researchers in BMC Genomics see advantages for capture-based Illumina sequencing and amplicon-based sequencing on the Nanopore instrument, depending on the situation or samples available.

LINE-1 Linked to Premature Aging Conditions

Researchers report in Science Translational Medicine that the accumulation of LINE-1 RNA contributes to premature aging conditions and that symptoms can be improved by targeting them.