Gene Expression System

CodeLink Gene Expression Bioarray System

Making it easier to understand biological systems

The CodeLink™ Gene Expression Bioarray System is an integrated platform that includes a range of high-quality, pre-arrayed oligonucleotide bioarrays, target preparation and bioarray processing reagents, easy-to-use processing tools, software, and optimized protocols.

CodeLink Gene Expression Bioarrays demonstrate superior results:

SENSITIVITY down to less than one transcript per cell enables detection of more low abundance genes.
SPECIFICITY up to 90% probe-to-target sequence identity leads to high levels of discrimination between homologous genes.
DYNAMIC RANGE > 3 orders of magnitude along with > 98% of probes within two-fold detectable change result in more usable data per bioarray.
Signal REPRODUCIBILITY with observed signal CVs of < 10% ensures minimal technical variation and enhanced discriminatory power

Proprietary 3-D surface technology for high sensitivity and specificity
The foundation of every CodeLink Bioarray is a proprietary 3-D aqueous gel matrix slide surface with 30-base oligonucleotide probes. Probe attachment is accomplished through covalent interaction between the amine-modified 5' end of the dispensed oligonucleotide and the activated functional group present in the gel matrix. The 3-D gel matrix provides an aqueous environment that holds the probe away from the surface of the slide, allowing for maximum interaction between probe and target. The solution phase hybridization kinetics , facilitated by the 3-D surface, results in higher probe specificity and sensitivity.

Fig.1 End-point attachment orients the DNA while the polymeric coating holds it away from the surface of the slide, making the DNA readily available for hybridization

Fig.2 The effect of 2 and 3 base mismatches across the entire length of the oligonucleotide probe illustrates the entire 30-mer participates in hybridization. Reference: Dorris et al., 2003

When the 3-D gel matrix is employed, mutation studies indicate that all bases of the CodeLink probes participate in hybridization and that the hybridization kinetics are very similar to those observed in solution-phase hybridization. The advantage of 30-mer probes is that they will have better discrimination and improved specificity over longer probes.

Industry leading sensitivity and dynamic range
The CodeLink Expression Bioarray System utilizes a one-color labeling and detection method based on biotin-labeled cRNA target prepared by linear amplification. PolyA(+) RNA within a total RNA population is primed for reverse transcription by a DNA oligonucleotide containing a T7 RNA polymerase promoter 5' to a d(T)₂₄ sequence. After second-strand cDNA synthesis, the cDNA serves as the template in an in vitro transcription reaction to produce the target cRNA. The in vitro transcription is performed in the presence of biotinylated nucleotides to label the target cRNA. This procedure produces a 1,000- to 5,000-fold linear amplification of the input polyA(+) RNA, often higher for many sources of total RNA.

The CodeLink Expression Assay Kit contains reagents for cDNA synthesis and in vitro transcription along with a set of well-characterized bacterial control mRNAs. The CodeLink assay can be monitored using these external RNA controls. Additionally, the bacterial control mRNAs can be used to estimate the dynamic range and sensitivity of the assay.

Using high-quality cRNA generated with the CodeLink assay protocol and validated instruments, the CodeLink Expression Bioarray System typically detects lower than one transcript per cell with as little as 100ng of total RNA starting material.

Probe selection and empirical validation for high specificity
Probe sequences for CodeLink Bioarrays are well-annotated and thoroughly screened in silico and tested under standard conditions to ensure optimal and accurate hybridization performance. Each probe is also empirically validated against multiple tissues to ensure optimal representation of the gene so that experimental results are biologically relevant.

Specificity is estimated at 90% probe homology to target sequences. This is important because a centrally located three base mismatch between probe and target sequences is sufficient to significantly reduce hybridization efficiency, allowing for discrimination of highly homologous gene sequences.

Fig. 3 CodeLink Bioarrays are highly specific. In this figure, each bar represents the average signal intensity from six bioarrays and 95% confidence intervals are displayed. Three-base mismatched 30-mer probes (3xMM) are not detected above noise. PM is perfect match, and MM is mismatch.

Quality controls and quality assurance for reproducible results

To ensure high quality and reproducible bioarrays, strict quality control of each process in the bioarray production are maintained. Before arraying begins, each oligonucleotide is quality assured by mass spectrophotometry, which guarantees that homogeneous, full-length probes are present. Probe homogeneity is crucial to obtaining reproducibility from array to array and from batch to batch.

Each presynthesized 30-base oligomer is deposited onto coated, standard-size slides. The deposition process is tightly controlled and closely monitored, and it takes place in a class 1000 clean room. Each and every completed bioarray is inspected for spot morphology and spot size. If a spot does not meet quality control standards, data analysis software automatically masks it in the final data set. If more than the allowed number of spots fail the quality check, then the entire bioarray is discarded.

After inspection, each batch is functionally validated to ensure performance specifications are met. Each CodeLink Bioarray is shipped with a unique Manufacturing System Report (MSR) that identifies those spots that failed quality control. At least 98% of the spots are present, hybridizable, and extractable.

Each bioarray is ensured to be of the highest quality and to give the most usable data possible. Signal variance for CodeLink Bioarrays is less than 10% with high levels of discrimination power. The rigorous manufacturing and quality control processes ensure excellent reproducibility and gives users higher confidence in their results.


Fig. 4 CodeLink Bioarrays deliver excellent signal reproducibility from array to array. In this figure, probe signals from the same target on two independent bioarrays are compared (outliers are included). The R value is 0.9986, which shows excellent correlation, linearity, and reproducibility.

	Typical Performance	Benefit
Sensitivity	Mass ratio <1:1 000 000 with bacterial spiking into cRNA and total RNA level	Detection of more low abundance transcripts (<0.3 copies per cell)
Dynamic range	Linear signal response across three orders of magnitude (0.05 pM – 50 pM)	More usable/accurate data across a broad range of gene expression levels
Specificity	Discrimination of >2 base mismatches	Better discrimination of highly homologous genes
Signal reproducibility	Typically <15% total median CV among production batches, all probes included; ~8% median CV among production batches, only probes above noise included	Reproducible differential expression results in every experiment
Minimum detectable fold change	>98% within two-fold change for replicates	Reduced noise for better detection of small changes in gene expression and more usable data per bioarray