mRNA was purified from K562 cells using the TurboCapture 384 mRNA Kit. While the isolated mRNA was immobilized in the wells of the TurboCapture plate, cDNA was synthesized. Using cDNA from 8 wells of the TurboCapture plate, duplex, real-time PCR of an apoptosis-related gene (Fas, p21, or p53) and GAPDH internal control was performed. Well-to-well reproducibility with regard to CT values was high, with CVs of <3%. All protocol steps were performed on a robotic workstation.
[A]Lysate containing poly A+ mRNA is added to a well containing immobilized oligo-dT. [B]Poly A+ mRNA hybridizes to the immobilized oligo-dT. [C]While the mRNA is hybridized, cDNA is synthesized using the immobilized oligo-dT as primer. The resulting cDNA is covalently linked to the well. (Alternatively, soluble primers can be used to synthesize soluble cDNA; not shown in figure.)
mRNA was purified from HEK 293 cells using the TurboCapture 96 mRNA Kit. While the isolated mRNA was immobilized in the wells of the TurboCapture plate, cDNA was synthesized. The resulting immobilized cDNA was subjected to 3 rounds of PCR, as indicated in the figure. The wells were washed 3 times with 10 mM Tris·Cl, pH 7.5 between each PCR. Wells 2 and 3 are duplicate samples. Wells 1 are negative controls in which cDNA synthesis was performed without mRNA. (Similar bands were obtained when PCRs of p53, p21, and β-actin were performed in different orders; data not shown.)
mRNA was purified from different numbers of K562 cells using the TurboCapture 96 mRNA Kit. While the isolated mRNA was immobilized in the wells of the TurboCapture plate, RT-PCR analysis of JunB was performed.
mRNA was purified from suspension cells (U937, K562, and Jurkat) using the TurboCapture 96 mRNA Kit. The isolated mRNA was eluted and then quantified.
DLD-1 colon carcinoma cells were transfected with different siRNAs targeting p21. After 72 hours, mRNA was purified using the TurboCapture 384 mRNA Kit. The expression levels of p21 relative to GAPDH were determined by real-time RT-PCR. Each bar of the graph represents the mean and standard deviation from 3 samples.