RNA was purified from HeLa cells using the AllPrep RNA/Protein Kit and reverse-transcribed. Serial dilutions of cDNA equivalent to 100 pg to 100 ng of total RNA were analyzed using a real-time RT-PCR assay for human GSK3B (glycogen synthase kinase 3 beta). Reactions were performed in duplicate. RNA purified using the RNeasy Mini Kit showed similar CT values and reproducibility (data not shown). NTC: No template control.
HeLa cells in a 96-well plate were transfected with a β-galactosidase-reporter plasmid. After 48 hours, protein was purified and β-galactosidase activity was assayed (yellow indicates enzyme activity). Protein was purified using either the AllPrep RNA/Protein Kit (AllPrep) or the RNeasy Mini Kit (cell lysate was passed through an RNeasy spin column, and protein was precipitated from the flow-through) (Other). Each column of wells shows triplicate results from one cell sample.
Total RNA was purified from HeLa cells and analyzed on the Agilent 2100 Bioanalyzer. The rRNA ratio (28S/18S) was 2.04. Total protein was purified from HeLa cells and analyzed by western blot. Analysis of duplicate protein samples with antibodies against Lamin (numclear protein) and TIM23 (mitochondrial protein) demonstrates that the total protein fraction contains proteins from subcellular compartments.