Components required for this protocol were included in the Two-Cycle Target Labelling and Control Reagents (Affymetrix, P/N 900494) and MEGAscript High Yield Transcription Kit, Ambion Inc, P/N 1334 with the exception of Ethanol (Sigma, E7023). The positive control Poly-A RNA is firstly diluted before spiking in with the sample RNA. Affymetrix supply a Eukaryotic Poly-A RNA Control Kit along with the Two- Cycle Target Labelling and Control Reagents. The kit is designed specifically to provide exogenous positive controls to monitor the entire GeneChip eukaryotic target labelling process. It is important to note that the Poly-A spikes were made up in non-stick RNase/ DNase free tubes (Ambion cat no. 12450(1.5ml)/ 12350(0.5ml)), which prevents the Poly-A spikes from sticking to the sides of the tubes and interfering with the final concentration of the positive controls.
The tube with first strand cDNA synthesis master mix (Table 2.5.3.1) was then flicked and centrifuged briefly. 2µl of this mix was added to 2µl of the 50ng/µl RNA sample. The tubes were flicked and centrifuged briefly before being incubated for 6 min at 70°C. They were then incubated for 2 min on ice and centrifuged briefly.
Reagents Amount
Poly-A RNA Control 2μl
T7-Oligo (dT) Primer (50 μM) 2μl
RNA + RNase-free water 16μl
Table 2.5.3.1: First-Strand cDNA Synthesis
The first cycle, first strand master mix was prepared as in Table 2.5.3.2. It is worth noting that Affymetrix suggest that if there are more than 2 samples that it is prudent to include extra to compensate for potential pipetting inaccuracy or solution lost during the process. The 5µl of first strand master mix was added to each sample, the tube gently flicked, briefly centrifuged and placed immediately at 42°C for 1hr and 72°C for 10 min before being placed on ice for 2 min.
Reagents Amount 5X 1st strand buffer 2μl DTT (0.1M) 1μl dNTP (10mM) 0.5μl RNase Inhibitor 0.5μl Superscript II 1µl Total 7μl
Table 2.5.3.2: 1st strand master mix
The first cycle second strand master mix was prepared by adding following reagents (Table 2.5.3.3). This 10µl mix was then added to each tube which were then flicked and centrifuged briefly before being placed at 16°C for 2hrs and 75°C for 10 min and then ice for 2 min.
Reagents Amount
RNase-free Water 4.8μl
dNTP (10mM) 0.4μl
MgCl2 (17.5mM) 4μl
E. coli DNA Polymerase 0.6μl
RNase H 0.2μl
Total Volume 10μl
Table 2.5.3.3: Second-strand master mix
The components for the first cycle IVT amplification (Ambion Megascript T7 kit) were assembled at room temperature. 5µl of each of the components ATP, CTP, UTP, GTP, enzyme mix and 10 x reaction buffers were added together for each sample included before being added to each sample. The tubes were gently flicked, centrifuged and placed at 37°C for 16hrs. Reagents Amount 10X Reaction Buffer 5μl CTP Solution 5μl GTP Solution 5μl 10X Reaction Buffer 5μl CTP Solution 5μl GTP Solution 5μl Total Volume 30μl
Table 2.5.3.4: First-Cycle, IVT Master Mix
The cRNA was then purified using the GeneChip Sample Cleanup module (Affymetrix, 900371) as recommended by the manufacturers instructions.
H2O and reading the quantity by using the NanoDrop (see section 2.5.2). 600ng is
required for the second cycle of the protocol. 2µl of freshly diluted random primers (3µg/µl) was added to each sample and the tubes were flicked, centrifuged briefly and placed at 70°C for 10 min before being placed on ice for 2 min.
The second cycle first strand mix was prepared by adding each reagent together as in Table 2.5.3.5 for each sample included. This 9µl was added to each sample before they were flicked, centrifuged and placed at 42°C for 1hr and ice for 2 min. After this, 1µl of RNase H was added to each sample before they were flicked, spun, and placed at 37°C for 20 min, 95°C for 5 min and ice for 2 min.
Reagents Amount
5X 1st Strand Reaction Mix 4μl
DTT, 0.1M 2μl
RNase Inhibitor 1μl
dNTP (10mM) 1μl
SuperScript II 1μl
Total Volume 9μl
Table 2.5.3.5: Second-Cycle, First-Strand Master Mix
4µl of a freshly prepared aliquot of T7 Oligo dT primer was added to each sample before they were flicked, centrifuged briefly and incubated at 70°C for 6 min and ice for 2 min. The second cycle second strand master mix was prepared by adding the following in a tube for each sample required (Table 2.5.3.6). This 125µl master mix was added to each sample before being flicked, centrifuged briefly and incubated for 2 hrs at 16°C. T4 DNA polymerase (2µl) was then added to each sample before incubating at 16°C for a further 10 min. After incubating the samples at 4°C for 2 min, they were immediately purified using the GeneChip Sample Cleanup module (Affymetrix, 900371) following the
Reagents Amount
RNase-free Water 88μl
5X 2nd Strand Reaction Mix 30μl
dNTP, 10mM 3μl
E. coli DNA Polymerase I 4μl
Total Volume 125μl
Table 2.5.3.6: Second-Cycle, Second-Strand Master Mix
All 12µl of cDNA were used for the second IVT step. The reagents required for this step were assembled at room temperature. The master-mix included 8µl RNase- free water, 4µl of IVT labelling buffer, 12µl IVT labelling NTP mix and 4µl labelling enzyme mix for each sample included. The 28µl volume was added to each sample before flicking, centrifuging briefly and incubating at 37°C for 16 hrs.
The biotin-labelled cRNA was purified using the GeneChip Sample Cleanup module (Affymetrix, 900371), as recommended by the manufacturers, and quantified using a NanoDrop. For quantification of cRNA when using total RNA as starting material, an adjusted cRNA yield needed to be calculated to reflect carryover of unlabeled total RNA. Using an estimate of 100% carryover, the formula below was used to determine adjusted cRNA yield:
Adjusted cRNA yield = RNAm - (total RNAi) (y)
Where, RNAm = amount of cRNA measured after IVT (µg), total RNAi = starting amount of total RNA (µg), y = fraction of cDNA reaction used in IVT
The final step of the entire process was to fragment 20µg of the biotin-labelled cRNA by adding 8µl of fragmentation buffer to 20µg of cRNA and bringing the total volume of the reaction to 40µl, so that the concentration of the cRNA is 0.5µg/µl. This mix was incubated for 35 min at 94°C. From the fragmented cRNA 30µl (=15µg) was hybridised to the Affymetrix U133-plus-2 chip.
2.5.3.1 Probe Array Scan
After staining and washing, the chips were scanned using an Affymetrix GeneChip Scanner 3000 (Affymetrix, 00-0186). The sample door on the scanner was opened and the probe array was inserted into the holder. Affymetrix GeneChip Operating Software (GCOS) runs all aspects of the array process, saving images of the scanned probe array in a data file (*.dat). GCOS automatically calculates the *.cel (Cell Intensity File) file from each *.dat file, which contains a single intensity value for each probe cell delineated by the grid (calculated by the Microarray Suite 5.0 (MAS5.0) algorithm) (ref- Affymetrix, I. Statistical Algorithms Description Document. 2002.
http://www.affymetrix.com/support/technical/whitepapers.affx ). The chip File (*.chp)
generated from the analysis of a probe array contains qualitative and quantitative analysis for every probe set. The report file (*.rpt) generated by GCOS summarizes the data quality information for a single experiment. The report is generated from the analysis output file (*.chp).
2.5.3.2 Quality assessment of Affymetrix microarray chips
The quality of the data generated with Affymetrix microarray chips was assessed based on different criteria including the scaling factor, background and noise levels, GAPDH 3‟/ 5‟ ratios and the % Present call.
Scaling factor: The scaling factor was the multiplication factor applied to each signal value on an array. A scaling factor of 1.0 indicates that the average array intensity was equal to the target intensity. Scaling factors vary across different samples and so there were no set guidelines for any particular sample type. However, Affymetrix advise that for replicates and comparisons involving a relatively small number of changes, the scaling/normalization factors (calculated by the global method) should be comparable among arrays. Larger discrepancies among scaling/normalization factors (e.g., three-fold
Background and noise levels: Although there are no official guidelines regarding background, Affymetrix has found that typical Average Background values range from 20 to 100 for arrays scanned with the GeneChip® Scanner 3000. Arrays being compared should ideally have comparable background values. A high background (over 60%) implies that impurities, such as cell debris and salts, are binding to the probe array in a non-specific manner, and that these substances are fluorescing at 570nm (the detection wavelength). This non-specific binding (noise) causes a low signal-to-noise ratio (SNR), meaning that transcripts present at very low levels in the sample may be incorrectly called as “Absent”. High background creates an overall loss of sensitivity in the experiment
GAPDH 3‟/ 5‟ ratios: β-actin and GAPDH are used to assess RNA sample and assay quality for the majority of GeneChip expression arrays. Specifically, the Signal values of the 3‟ probe sets for β-actin and GAPDH are compared to the Signal values of the corresponding 5‟ probe sets. The ratio of the 3‟ probe set to the 5‟ probe set is generally no more than 3 for the 1-cycle assay. A high 3‟ to 5‟ ratio may indicate degraded RNA or inefficient transcription of ds cDNA or biotinylated cRNA. 3‟ to 5‟ ratios for internal controls are displayed in the Expression Report (.rpt) file.
%Present call: The number of probe sets called “Present” relative to the total number of probe sets on the array is displayed as a percentage in the Expression Report (.rpt) file. Percent present (%P) values depend on multiple factors including cell/tissue type, biological or environmental stimuli, probe array type, and overall quality of RNA. Replicate samples should have similar %P values. Extremely low %P values are a possible indication of poor sample quality. In practice, % present calls averaged between 40-60% for cell line RNA and 15-25% for clinical specimens.