Focus: Labeled cDNA Purification for Microarray Analysis
The ChipShot™ Membrane Clean-Up System: A Fast Simple Technique for Efficient Recovery of Highly Purified cDNA
We show that the ChipShot™ Labeling Systems when used in combination with the ChipShot™ Membrane Clean-Up System provide superior-quality labeled cDNAs for microarray analysis.
Nadine Nassif, M.S., Promega Corporation
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 is included as the cDNA clean-up component of both the Pronto!™ Plus Direct (Cat.# 40055, 40056) and the Pronto!™ Plus Indirect (Cat.# 40075, 40076) Systems. Compared to previous clean-up methods that relied on silica beads or particles for purification, the new 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.
| A. Direct Labeling of cDNA from Total RNA. | ||||
|---|---|---|---|---|
| cDNA yield (ng) | Dye Incorporated (pmol) | FOI | ||
| Cy®3 | ||||
| ChipShot™ Membrane Clean Up (n = 8) | 2,128 ± 101 | 193 ± 10 | 29 ± 0 | |
| Cy®5 | ||||
| ChipShot™ Membrane Clean Up (n = 8) | 2,132 ± 88 | 140 ± 4 | 21 ± 0 | |
| B. Indirect Labeling of cDNA from Total RNA. | ||||
| ng cDNA before dye conjugation | ng cDNA after dye conjugation | Dye Incorporated (pmol) | FOI | |
| Cy®3 | ||||
| ChipShot™ Membrane Clean Up (n = 6) | 3,633 ± 287 | 3,171 ± 213 | 174 ± 11 | 18 ± 2 |
| Cy®5 | ||||
| ChipShot™ Membrane Clean Up (n = 6) | 3,619 ± 204 | 3,206 ± 140 | 249 ± 14 | 25 ± 2 |
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.
Figure 1. cDNA yield achieved using the ChipShot™ Membrane Clean-Up System compared to competing systems. Sixteen Cy®3-labeled cDNA reactions were synthesized using the ChipShot™ Direct Labeling System. Following RNase treatment, the reactions were pooled, then redivided into sixteen 40μl aliquots, eliminating any sample-to-sample variation prior to purification. Replicate samples were purified with the ChipShot™ Membrane Cleanup System; post-purification yields are compared to several alternate purification systems (n = 4 for each method 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(a) 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.
Figure 3. Highly pure cDNA produced using the ChipShot™ Systems. Fluorescently labeled cDNA was generated using the ChipShot™ Direct Labeling System (lanes 1 and 2) and the ChipShot™ Indirect Labeling System (lanes 3 and 4). 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 (lanes 5 and 7) and 0.5pmol Cy®-dCTP (lanes 6 and 8) are shown in comparison, demonstrating the high level of purity achieved when using the ChipShot™ Membrane Cleanup System. Lane M, ALFexpress® Sizer™ 50-500 (Amersham Biosciences Cat.# 27-4539-01). The gel was scanned on a 9410 Typhoon® Imager (Amersham Biosciences).
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.
Figure 4. Array image following hybridization with Cy®-labeled cDNA purified using the ChipShot™ Membrane Clean Up System. Cy®3-labeled cDNA from 293T mRNA and Cy®5-labeled cDNA from HeLa mRNA was synthesized using the ChipShot™ Direct Labeling System and purified using the ChipShot™ Membrane Cleanup System. The cDNA samples were hybridized to custom 4K arrays (provided by Corning, Inc.) 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.
(a)The PCR process is covered by patents issued and applicable in certain countries*. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process.*In Europe, effective March 28, 2006, European Pat. Nos. 201,184 and 200,362, will expire. In the U.S., the patents covering the foundational PCR process expired on March 29, 2005.
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