SpotQC

Microarrays are widely used to study gene expression on a genome-wide scale. Many facilities print their own microarray slides using cDNA/PCR products or oligonucleotides. Quality control is essential to ensure data integrity; it is crucial to verify that all spots are present on printed slides - i.e., that nucleic acid was actually deposited for all spots on all slides produced in a print run. Spot drop out is a commonly encountered problem in printed arrays that can lead to false negatives.

A perfect QC method would employ hybridization of complementary targets specific to each probe spotted on a slide. However, this approach is too expensive and complex for general use. One commonly employed method to QC printed arrays is to hybridize representative slides from a print run with a dye-labeled oligo. Sequence variation between individual spots leads to huge variation in signal intensity between spots and limits utility of this method, especially for oligonucleotide arrays.

IDT has developed a Cy3^TM dye-labeled oligonucleotide with improved thermodynamics that will hybridize with greater uniformity to the wide variety of sequences employed in large-scale microarrays. Our Cy3^TM-detector oligo used with our optimized hybridization solution permits rapid, precise QC of printed microarray slides. Hybridization, wash, and image acquisition can be done in under an hour. While the benefit of our new formulation is most evident with oligonucleotide arrays, SpotQC can also be used with superior results for cDNA/PCR product arrays. SpotQC has improved sensitivity, detects a greater range of oligo-probe lengths and GC compositions, and more accurately assesses the spotted probe's availability for hybridization vs. traditional random fluorescent primers. Biotin SpotQC employs a biotin-labeled detector oligo which permits the use of enzymatic-based chemiluminescent or colorimetric detection options for the QC assay.

IDT's SpotQC Image vs. Cellular RNA Hyb Results on a High-Density cDNA Array. The left image was obtained from a 1 hour QC hybridization using the IDT Cy3-SpotQC detector oligo. The right image was obtained from an overnight hybridization of a sister slide (same print run) done using Cy3 ^TM /Cy5 ^TM -labeled cellular RNA. The utility of SpotQC to distinguish between true negatives and false negatives is demonstrated. White arrows indicate examples of real negatives where the absence of experimental hyb signal is due to absent (or very low) mRNA target and not a printing defect. Other missing spots can be found which are absent in both the QC image and the cellular RNA hyb image, which are spot drop outs; these data points can now be eliminated from the final gene expression analysis done on the experimental array and thereby prevent false negative results. The SpotQC hybridization image is 5-micron resolution Cy3 ^TM scan using a ScanArray® 5000 (Perkin Elmer) at 70/70 laser (power/gain) settings. (Pseudo-color scale: black<blue<green<yellow<orange<red<white). The cellular RNA hybridization image is 5-micron resolution Cy3 ^TM /Cy5 ^TM composite using a GenePix ^TM (Axon) scanner. (Cy3 ^TM , green; Cy5 ^TM , red; Cy3 ^TM /Cy5 ^TM , yellow).

Cy3^TM SpotQC Detector oligo

Detector oligo is shipped with a complimentary tube of 1X Hybridization Buffer (500 µl). The quantity is sufficient to analyze 10 arrays using large 24x60mm cover slips or up to 30 arrays using small 22x22mm cover slips.

Biotin SpotQC Detector oligo

IDT’s Bio-SpotQC is a biotinylated version of SpotQC, and has been developed to precisely QC printed microarray slides utilizing secondary fluorescent, chemiluminescent, or colorimetric hybridization detection assays. (Secondary fluorescent, chemiluminescent, and/or colorimetric detection assay reagents NOT included.)

Detector oligo is shipped with a complimentary tube of 1X Hybridization Buffer (500 µl). The quantity is sufficient to analyze 10 arrays using large 24x60mm cover slips or up to 30 arrays using small 22x22mm cover slips.

Epoxide Spotting Buffer (ESB)

IDT’s Epoxide Spotting Buffer is an optimized microarray printing solution that dramatically improves spot morphology and uniformity, both of which are essential for producing high-quality arrays and reliable data. ESB is optimized for spotting either amine-modified or I-Linker^TM-modified oligo probes on epoxy surface slides. The combination of IDT’s I-Linker^TM modification and ESB results in a 2-fold increase in microarray spot hybridization signal over traditional amine-modified oligo probes printed on an epoxide surface. ESB is designed for reduced evaporation and is suitable for either short or overnight microarray print runs.