Our protocol on the simultaneous extraction of DNA, RNA, and proteins from frozen hiPSCs have been published at Current Protocol. This work was a collaboration with Jeffrey Zhang, David Paik, and Yan Zhuge at Joseph Wu's laboratory at Stanford Cardiovascular institute.
Human induced pluripotent stem cells have become an important cellular model to understand disease mechanism and identify cellular targets. As technologies mature and hiPSC lines from donors of different genetic backgrounds proliferate there has been a need for persistent resources to provide standardized lines to researchers. The Stanford Cardiovascular Institute for instance currently hosts one of the largest biorepositories for hiPSC lines related to cardiovascular diseases, with over 1000 distinct lines in its collection available for research use. Other biorepositories and commercial vendors also exist that allow individual labs to request established hiPSC cell lines without having to devote resources to generate and validate the hiPSC lines de novo.
In this protocol we evaluated the quantity or quality of extracting various biomolecules directly from one to two vials of cryopreserved hiPSCs containing a few million live cells, such as would be typically received from an hiPSC repository or vendor. This protocol would be important for an individual lab to genotype or validate received hiPSCs, or to perform experiments to assess the gene expression profiles of different hiPSC lines. We adopted the Qiagen AllPrep kit which can simultaneously extract DNA, RNA, and proteins from one same sample, and then evaluated the effects of different storage factors on total yield and DNA quality.
We applied this protocol to over 700 different hiPSC lines and found that it can extract sufficient amount of DNA and RNA for next-generation sequencing purposes from 1–2 vials of cryopreserved hiPSCs. The extracted genomic DNA quality was high with minimal degradation, and importantly good yield was recoverable from hiPSCs that were cryopreserved for up to four years.
Please feel free to go over to Current Protocols in Stem Cell Biology for the detailed protocol and let us know what you think in the comments section below. A simplified print-ready version is also hosted on the Protocols section of our website.