Introduction
The ChipShot™ Membrane Clean-Up System provides a method for rapid purification of dye-labeled cDNA. The system uses a newly developed silica membrane spin column for affinity purification of the labeled cDNA. This method effectively removes unincorporated nucleotides, primers and free dye, while yielding highly efficient cDNA recovery. The ChipShot™ Membrane Clean-Up System was originally included as the cDNA clean-up component in both the Pronto!™ Plus Direct and Indirect Systems. Compared to previous clean-up methods that relied on silica beads or particles for purification, the ChipShot™ Membrane Clean-Up System provides a faster, simpler technique for efficient recovery of purified cDNA. Used in combination, the ChipShot™ Labeling Systems and ChipShot™ Membrane Clean-Up System provide superior methods for efficiently synthesizing labeled, high-purity cDNA for use in microarray experiments.
Yield and Labeling Efficiency
Total RNA from a commercial source was used as the template for cDNA synthesis with the ChipShot™ Direct Labeling System (Table 1, Panel A) and the ChipShot™ Indirect Labeling System (Table 1, Panel B). For the ChipShot™ Indirect Labeling System, cDNA yield is shown for both the post-synthesis and post-conjugation purifications, demonstrating highly efficient cDNA recovery between purification steps. Yield and frequency of incorporation (FOI) were calculated using absorbance values at 260, 550, and 650nm. The number of replicate reactions per sample is shown in parentheses; values include standard deviation.
Table 1. Yiled and Labeling Efficiency of Dye-Labeled cDNA Using the ChipShot Membrane Clean-Up System. 
Achieve Better Yield with the ChipShot™ Membrane Clean-Up System
We synthesized 16 Cy®3-labeled cDNAs using the ChipShot™ Direct Labeling System. Following RNase treatment, we pooled the reactions and then redivided them into sixteen 40μl aliquots, eliminating any sample-to-sample variation before purification. Replicate samples were purified using the ChipShot™ Membrane Clean Up System as described in Technical Manual #TM243. Post-purification yields are compared to several alternate purification systems in Figure 1. Four replicate samples were purified with each method. The ChipShot™ Membrane System delivered higher yield than the competing systems tested.
Remove Unincorporated Nucleotides and Primers While Retaining Purified cDNA
Unlabeled and fluorescently labeled oligonucleotides ranging from 15- to 75-mers were quantitated and then applied to ChipShot™ columns. Following purification using the ChipShot™ Membrane Clean-Up System, we quantitated the oligonucleotides a second time to assess the efficiency of the ChipShot™ membrane at retaining differently sized nucleic acids. We performed a similar study using fluorescent PCR products. The results shown in Figure 2 demonstrate the effectiveness of the ChipShot™ membrane at removing unincorporated nucleotides and primers while efficiently retaining purified cDNA.
Figure 2. Retention of nucleic acid fragments as a function of size, using the ChipShot™ Membrane Clean-Up System.
Pre-purified oligonucleotides (15-, 30-, 45-, 60-, and 75-mer in length) were quantitated and applied to ChipShot™ columns. Following purification with ChipShot™ Membrane Clean-Up System, the oligonucleotides were requantitated to assess the efficiency of the ChipShot™ membrane at retaining differently sized nucleic acids. Both unlabeled and fluorescently labeled oligonucleotides were tested. Similarly, retention of prepurified fluorescent PCR products (60bp, 80bp, 100bp, 200bp, 300bp, and 400bp in length) was also assessed.
Obtain Highly Pure cDNA
We generated fluorescently labeled cDNA using the ChipShot™ Direct Labeling System and the ChipShot™ Indirect Labeling System (Figure 3). The labeled cDNA samples were purified using the ChipShot™ Membrane Clean-Up System. Following purification, 1pmol of labeled cDNA was resolved on a 5% Long Ranger® denaturing acrylamide gel. Migration of 0.5pmol Cy-Dye™ NHS ester dye and 0.5pmol Cy®-dCTP are shown in comparison, demonstrating the high level of purity achieved when using the ChipShot™ Membrane Clean-Up System.
Produce Excellent Microarray Data Using cDNA Labeled and Purified with ChipShot™ Systems
We synthesized Cy®3-labeled cDNA from 293T mRNA and Cy®5-labeled cDNA from HeLa mRNA using the ChipShot™ Direct Labeling System, and purified using the ChipShot™ Membrane Clean-Up System. The cDNA samples were hybridized to custom 4K arrays using the Pronto!™ Plus Direct System. Data were acquired using a Genepix® 4000B scanner (Axon Instruments, Inc.); a representative subgrid of the array is shown in Figure 4. Strong signal and low background is achieved with this system, demonstrating the high quality of the purified cDNA.