The Affymetrix GeneChip® Whole Transcript (WT) Sense Target Labeling Assay is designed to generate amplified and biotinylated sense-strand DNA targets from the entire expressed genome without bias. The 1μg Total RNA Labeling Protocol starts with a ribosomal RNA (rRNA) reduction procedure where the 28S and 18S rRNA population is significantly reduced from the total RNA sample, minimizing the background and thereby increasing the array detection sensitivity and specificity. The rRNA reduction becomes critical when a user is interested in high-sensitivity analysis of expression levels for both genes and exons using the GeneChip® Exon 1.0 ST Arrays. This is because exon probe sets contain a smaller number of probes and in some cases selection of those probes is constrained by the limited size of the probe selection region. Therefore, it is imperative to use the high-sensitivity assay for optimal performance. However, the analysis of the gene level benefits from a larger number of high-quality probes selected from the entire transcript, thus the advantage of the additional rRNA reduction step is reduced. Therefore, the 100ng Total RNA Labeling Protocol is acceptable for use with the GeneChip® Gene 1.0 ST Arrays. Experimental turn-around times are usually three to four business days.
From total RNA, double-stranded cDNA is synthesized with random hexamers tagged with a T7 promoter sequence. The double-stranded cDNA is subsequently used as a template and amplified by T7 RNA polymerase, producing many copies of antisense cRNA. In the second cycle of cDNA synthesis, random hexamers are used to prune reverse transcription of the cRNA from the first cycle to produce single-stranded DNA in the sense orientation. In order to reproducibly fragment the single-stranded DNA and improve the robustness of the assay, a novel approach is utilized where dUTP is incorporated in the DNA during the second-cycle, first-strand reverse transcription reaction. This single-stranded DNA sample is then treated with a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) that specifically recognizes the unnatural dUTP residues and breaks the DNA strand. The DNA is labeled by terminal deoxynucleotidyl transferase (TdT) with the Affymetrix® proprietary DNA Labeling Reagent that is covalently linked to biotin. Following the recommended procedures, sufficient target is anticipated to be generated for hybridization to a single array.
IsolationThe analysis begins with total RNA that is provided by an investigator. In our practice, isolating total RNA from mammalian tissue using a reagent such as TRIzol followed by additional purification using RNeasy from QIAGEN yields RNA that produces satisfactory microarray data. In addition, successful isolation of total RNA from mammalian cells has been purified and eluted using RNeasy. Modifications of these methods, as well as other methods, are also known to produce suitable total RNA. Please contact our core facility if you are uncertain as to whether an RNA extraction method that you are considering is suitable.
QuantityInvestigators need to provide documentation of sample quantity before experiments begin. All sample quantities are rechecked on the NanoDrop 1000 Spectrophotometer. Once the quantity has been rechecked and verified for each sample, your experiment will begin. If quantity is an issue for any sample, the investigator will be notified immediately to discuss the next course of action.
The 1 or 2μg Total RNA Labeling Protocol requires either 1 or 2μg of total RNA as starting material, where the concentration should not fall below 0.31μg/μL for 1μg or 0.62μg/μL for 2μg. We recommend samples concentrated at 1μg/μL for simplicity. Please provide an extra 2ug of total RNA in addition to the experimental quantity submitted in order to account for the protocol checkpoints and any concentration variations, should your sample concentrations be off.
The 100 to 300ng Total RNA Labeling Protocol requires 100 to 300ng of total RNA as starting material, which should be suspended in a maximum of 3μL of solution in volume. We recommend samples concentrated at 100ng/µL for simplicity. Please provide an extra 200ng of total RNA in addition to the experimental quantity submitted in order to account for the protocol checkpoints and any concentration variations, should your sample concentrations be off.
QualityThe quality of the RNA is essential to the overall success of a gene expression array. The Genomics Core Facility requires an agarose gel picture of the submitted samples that clearly defines the prominent 28S and 18S rRNA bands for each sample. Investigators have to provide this documentation of sample quality before experiments begin. The spectrophotometric absorbances from the NanoDrop 1000 are checked at A260, A280, and A230 nm for each sample. The A260/280 ratios for each sample should be close to 2.0 for pure RNA samples (ratios between 1.7 and 2.1 are acceptable), while the A260/230 ratios for each sample should be between 1.8 and 2.2 for high-quality RNA. In addition, all sample qualities are rechecked on the Agilent 2100 Bioanalyzer. Once the quality has been verified for each sample, your experiment will begin. If quality is an issue for any sample, then the investigator will be notified immediately to discuss the next course of action.