We supply shRNAs and ORFs from our libraries to Einstein, Montefiore, and Yeshiva University researchers in these formats:
- Bacterial glycerol stocks
- Plasmid DNA
- Lentivirus, either as viral supernatant (200 µl >106 TU/ml) or concentrated viral stock (50 µl at >108 TU/ml)
Note: The shRNA Core Facility does not supply packaging plasmids or cells for generation of lentivirus. Upon request, we can provide you with information on sources for these materials.
For researchers at other New York institutions, we supply shRNAs as viral supernatant (106 TU/ml) only.
If you are interested in using shRNAs for a screening application, please contact us to discuss options. There are a number of ways that the shRNA Facility can help:
- We can prepare custom arrayed libraries of shRNAs in lentiviral format for screening purposes.
- Pooled library screens are available using the Decode and DECIPHER libraries. This format is appropriate for enrichment or dropout screens where a selection for the desired phenotype can be applied. Hits can be identified by next-generation sequencing.
- We can also prepare custom pooled libraries containing subsets of the GIPZ or TRC libraries.
The shRNA Core Facility has the following equipment available for assays:
- EnVision Multiplate Reader
- Operetta High-Content System
Assessment of knockdown
A critical part of an RNAi experiment is confirmation of knockdown. In some cases this can be accomplished by Western blot or other analysis of the protein product. However, in many cases, quantitation at the level of the mRNA is necessary. The shRNA Facility can assess knockdown of targets at the mRNA level using the branched DNA assay. This assay is based on hybridization of the mRNA species of interest in a cell lysate to a specific set of primers, with signal amplification accomplished through use of a branched DNA reagent.
We are available to consult on RNAi experiments and offer our expertise on experimental design and data analysis.