Single Cell Sample Prep for Genetic Analysis
Efficient lysis of single cells for PCR is not necessarily a challenging aspect of sample prep, in particular with mamalliam cells - which do tend to be the focus for single cell analysis.
However lysis is not sufficient for reliable and reproducible PCR and RT-qPCR from single cells. Heat lysis of single cells results in denatured protein getting stuck to the nucleic acid template which adversely effects the processivity of DNA polymerases and, even more so, RT-polymerases.
What is required is removal of the protein material from the nucleic acid templates to allow free and ready amplification or reverse transcription.
The ZyGEM (Hamilton, NZ) reagents for sample prep are ideally suited to this purpose and have been licensed to a number of companies for inclusion in their kits, such as the world leading Picoplex WGA kits from Rubicon. ZyGEM's latest release, RNAGEM, delivers linear yields of DNA and RNA from 1-150,000 cells. The kit is easily scalable and extractions can be done in 1ul volumes.
Obviously, when handling single cells it is critically important that there is no attempt to purifiy the nucleic acids as any attempt to purifiy nucleic acids from ANY sample type results in template loss. Generally this is not a problem when working with large amounts of sample, but with single cells this will compromise the data. The RNAGEM reagents are totally compatible with RT and PCR reagents and there is no requirement to purify the template post extraction.