The normalization of esiRNA products.

<p><b>A.</b> A range of initial amounts of DNA template were assayed (100, 10, 1, and 0.1 ng, each in 5 µL in volume). After the immobilization and transcription steps, the variation of transcription products was within 20%. The final esiRNA products had a variation of less than 10% when the initial amount of DNA templates was in the range of 0.1–100 ng. <b>B</b>. Eight esiRNA products were manufactured in parallel. The standard deviation among these eight products was approximately 3%.</p

Manufacture of esiRNA by means of a magnetic bead-integrated chip.

<p><b>A.</b> The schematic diagram shows the chip composed of a microwell array and magnetic beads coated with streptavidin. <b>B.</b> Large-scale manufacture of esiRNAs can be divided into three steps: target amplification and immobilization, transcription, and enzymatic digestion. <b>C.</b> Confirmation of PCR reactions using biotinylated or non-biotinylated primers. Amplification products (left), transcription products (middle), or esiRNA products (right) are detected only when biotinylated DNA primers are used. B: biotinylated primers.</p

Screen and validation of tyrosine kinase genes capable of regulating the cell migration. A.

<p>Self-assembled cell microarray screen of genes capable of regulating the migration of Hela cells by use of esiRNA tyrosine kinase library. <b>B.</b> Validation of the esiRNA screen results by use of siRNAs and transwell assay.</p

The silencing specificity and efficiency of esiRNAs. A.

<p>Fifteen GFP esiRNAs generated in parallel potently inhibited the fluorescent signal in cells cotransfected with GFP-encoding vectors; in contrast, five PLAU esiRNAs had no effect on the fluorescent intensity compared to control experiments. <b>B. & C.</b> Quantitative analysis of the silencing efficiency of esiRNA products. The qRT-PCR results showed that esiRNAs manufactured on the magnetic beads can efficiently inhibit expression levels of the respective genes. A western blot assay showed that esiRNAs targeting TP53, TGFB1, PLAU, or TGFBR2 inhibited therespective protein expression levels by up to approximately 50%.</p