We supply shRNAs and ORFs from our libraries to Einstein, Montefiore, and Yeshiva University researchers in these formats:
- Lentivirus, either as viral supernatant (200 µl >106 TU/ml) or concentrated viral stock (50 µl at >108 TU/ml)
- Plasmid DNA
- Bacterial glycerol stocks
We can also perform transductions into your cells and generate stable shRNA-expressing cell lines.
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 several other New York City institutions, we supply shRNAs as viral supernatant (106 TU/ml) only. Please contact us for details.
If you are interested in modulating gene expression in a screening context, please contact us to discuss options. We can perform screens in two formats:
- We can prepare custom arrayed lentiviral libraries for screening. The readout can be plate-reader-based or image-based.
- We can perform screens with pooled lentiviral libraries. The pooled format is appropriate for enrichment or dropout screens where a selection for the desired phenotype can be applied. Hits are identified by next-generation sequencing.
Screens can be performed with three different gene modulation approaches:
- Knockdown screens (shRNA)
- Knockout screens (CRISPR)
- Overexpression screens (ORF)
The shRNA Core Facility has the following equipment available for assays:
- EnVision Multiplate Reader: for fluorescence, luminescence, absorbance, and other 96- or 384-well based plate reader assays.
- Operetta High-Content System: for image-based readouts in 96-well, 384-well, and other formats. Analysis software permits quantitation of a wide variety of cell parameters.
We can generate stable knockout cell lines for your genes of interest using CRISPR/Cas9.
ORF and cDNA reagents
We have several collections of human and mouse open reading frames. We can supply these as glycerol stocks, plasmid DNA, or lentivirus (for those that are in lentiviral vectors). We can also generate stable overexpressing cell lines from constructs in the lentiviral collections.
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.