The Ambion Cells-to-cDNA II Kit is designed to streamline the process of gene expression analysis by making cDNA directly from cell lysates without a RNA isolation step. According to Ambion, the kit reduces the time required to obtain quality cDNA from cultured cells, allowing RT-PCR analysis for a large number of samples. In addition, a relatively small number of cells are required for each sample. Adherent cells may be directly lysed if grown in 96-well plates or may be detached and washed in PBS prior to lysis if grown in larger vessels. Suspension cells should be washed with PBS unless grown in low serum conditions. Lysis is performed by mixing the cells with ice-cold lysis buffer and heating at 75
oC for 10-15 min. The protocol specifies 15-min DNase I treatment at 37
oC and 5-min heat inactivation of the DNase at 75
o, prior to cDNA synthesis. Protocols and materials (except for the thermostable DNA polymerase) for one-step and two-step RT-PCR are provided.
Ambion stresses the importance of a pilot experiment to optimize the number of cells per sample. If too many cells are used, there may be insuffient inhibition of the RNases in the sample by the lysis buffer. Detailed instructions, RNA controls and a primer pair for the pilot experiment are provided with the kit. The kit seems to be well-designed for high throughput assays of homologous samples. It has been previously tested with HeLa, CHO, COS-7, J558 and MCF-7 cell lines and should provide adequate sensitivity for analysis since GAPDH (a highly abundant house-keeping gene) could be detected in concentrations as low as 5 cell equivalents.
I have tried this kit with mouse lymphocyte cell line, EL4 and human epithelial cell line, A549. I am left with mixed feelings about the kit. On one hand, the pilot experiments worked well and the kit seemed to save time for cDNA extraction from the cells. On the other hand, I observed relatively low sensitivity and high variability between the samples. The moderately abundant house-keeping gene, HPRT, usually had cycle threshold values (Ct) of around 27 in real-time RT-PCR assays using the Cells-to-cDNA Kit, while traditional protocols (purified RNA to cDNA) had a Ct of around 20 (128-fold decreased sensitivity). In addition, the Ct values obtained from identical samples varied by as a much as 3 cycles for Cells-to-cDNA-based assays compared to less than 1 cycle variability for assays staring from purified RNA. I drew the following conclusions from my observations: 1. Ambion’s Cells-to-Signal Kit may be better for quantitative RT-PCR analyses if genomic DNA does not pose a problem (i.e. primers span introns and do not amplify from genomic DNA). 2. Standard protocols for RNA isolation and DNase treatment should be used in applications where genomic DNA may interfere (i.e. intronless genes and processed pseudogenes are present). 3. A competitor’s kit (i.e. Invitrogen’s SuperScript CellsDirect cDNA synthesis kit) might be worth testing.
Timur Yarovinsky, PhD
Associate Research Scientist
The University of Iowa