Quantitation sh/siRNA-induced Knockdown by qRT-PCR and WB
There are many algorithms that predict most effective siRNA and shRNA sequences, however experimental in vitro test of such sequences usually demonstrate that less than 30% of the constructs tested provide high level of gene silencing (over 80% target mRNA reduction). While testing si/shRNA silencing efficacy, it is also important to take into consideration overall transfection efficiency of certain cell lines and use of associated positive and negative controls.
Altogen Labs provides complete RNAi gene silencing services, including design, synthesis of siRNA and shRNA, validation of gene silencing, and functional testing. Quantification of reduction in protein expression and mRNA expression verify the silencing effects of siRNA transient transfection and/or shRNA incorporation and stable expression. Altogen Labs offer siRNA design, synthesis, purification and functional testing to measure percent (%) of mRNA knockdown using cell line of interest (over 120 cell lines available in-house).
Standard RNAi gene silencing services include:
Design, synthesis, HPLC purification, and annealing of 5 different siRNA molecules targeting gene of interest. siRNAs are tested for gene silencing potency. Two out of five siRNAs are finally selected (siRNAs are guaranteed to provide at least 80% mRNA expression knockdown). Validation of gene silencing (RNAi) effect performed by real-time qRT-PCR (mRNA expression) or Western Blot (protein expression). Client provides the gene name and accession number of the target gene and we’ll do the rest.
shRNA gene silencing using custom cell lines
Another option for the client is to provide target gene name (and accession number) and mail the cells to Altogen Labs, and our lab team does the rest. Altogen’s shRNA knockdown service includes design, construction, and cloning of shRNA into commercially available vectors, design and synthesis of the SYBR-labeled qPCR primers for quantitation of target gene expression, shRNA transfection into the cell line supplied by the customer. Gene expression analysis is performed by qPCR at 24, 48, and 72 hr post-transfection. Altogen Labs guarantees 80% or more inhibition of target gene expression.
Please send the details of your project, and we will provide an accurate quote and timeline estimate (contact e-mail firstname.lastname@example.org or call Altogen Labs technical support: 512-433-6177).