RNA Extractions using Rneasy Lipid Tissue kit RNA extraction using QIAzol: 1. A 4 ml of QIAzol reagent. Do not allow tissue to thaw before addition of QIAzol reagent. 2. Homogenize with Polytron for 60 seconds at setting 4. 3. Let samples sit at RT for 5 minutes. 4. Add 1.0 ml chloroform per sample. 5. Cap tubes securely and shake vigorously by hand for 15 seconds. 6. Let sit at RT 3 minutes. 7. Centrifuge @ 5,000 x g for 15 minutes at 4oC. 8. Transfer the aqueous phase to an eppendorf tube (1 ml/tube). 9. Add 1 volume of 70% ethnol (about 3 ml) mix thoroughly by vortexing. Continue without delay with Rneasy Midi columns. B. RNeasy Midi Kit 1. Add 4 ml of the sample onto midi Spin column. Close the tube and centrifuge at 5000 x g for 5 min at room temp. Discard the flowthrough. 2. Repeat the step 1 with remainder of the sample. Discard the flowthrough. 3. Add 4.0 ml of Buffer RW1 onto the spin column and centrifuge at 5000 x g for 5 min to wash the column. Discard the flowthrough. 4. Add 2.5 ml of RPE Buffer to the spin column. Close the tube and centrifuge at 5000 x g for 2 min. Discard the flowthrough. 5. Add another 2.5 ml of RPE Buffer to the spin column. Close the tube and centrifuge at 5000 x g for 5 min. Discard the flowthrough. 6. Place column in fresh tube. Add 150 ìl of Rnase free water to elute total RNA. Let the column and the tubes stand for 10 min at room temp and then centrifuge at 5000 x g for 5 min. 7. Repeat the elution with the flow through (150 ìl). 8. Take Ods at 260 and 280 nm on the scanning spectrophotometer in AFFY core lab.
Label
biotin
Label protocol
The MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kit made by Ambion was used. The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter using ArrayScript™ a reverse transcriptase (RT). The cDNA then undergoes second strand synthesis and clean-up to become a template for in vitro transcription (IVT) in a reaction containing biotin-modified UTP and T7 RNA polymerase. Once purified, the biotin-labeled aRNA is suitable for use on microarray gene expression systems designed for biotin-labeled antisense RNA samples. http://www.ambion.com/techlib/prot/fm_1791.pdf
Hybridization protocol
Hybridization is performed overnight in a temperature-controlled shaking incubator. Post-hybridization processing includes a stringent wash to remove unbound and nonspecifically hybridized target molecules, a staining step with a Cy™5-Streptavidin conjugate, and several non-stringent washing steps to remove unbound conjugate. Following a final rinse, the bioarrays are dried by centrifugation. http://www.appliedmicroarrays.com/pdf/codelink_bioarray_processing_protocol.pdf
Scan protocol
Arrays are scanned using GenePix 4000B scanner and analyzed using Amersham CodeLink Expression Analysis v.4.0 software. Codelink Bioarray QC (Rat) Bioarrays are examined by eye, and by a physical mapping of the flagged spots to qualitatively assess whether or not the image has any obvious flaws: high background, smears, scratches, etc. Codelink EXP v4.1 software is used to analyze acquired bioarray images during scanning. The analysis generates both raw and median normalized intensities. Also, the software is used as a primary point for quality control checks. The Bioarray QC report details several adjustable pass/fail criteria. Listed below are descriptions, and the threshold cutoffs we have selected for chip hybridization failures. Note that our settings are different than the default settings. If any chip statistic is found lower than the threshold setting, the chip is failed. The failed chip data is still made available, but with a notation on the specific failure. The failed chip data can be more closely scrutinized using a variety of measurement methods, and may still be effectively used in an analysis. % Total Spots Flagged Exceeds: 40 default(50) Spots that exhibit background contamination, irregular shape, low signal, or saturated signal. % Near Background Exceeds: 40 default(50) Spots where spot mean intensity/local background < 1. We have chosen 40% based on a large quantity of chips. % Contaminated Exceeds: 3 default(10) Spots that have background contamination. The background pixel dispersion has high contrast as seen in the expected mean and median statistics, or the local background is very high, compared to global background. We typically see less than 3% of spots flagged contaminated. % Irregular Exceeds: 3 default(5) Spots where percentage of the difference between maximum and minimum diameter of the spot measured in different directions exceeds a set value. We typically see less than 3% of spots flagged irregular. % Saturated Exceeds: 5 default(5) The percent of spots that have saturated signals. % Excluded MSR/User Exceeds: 2 default(2) Spots that did not pass manufacturing QC are masked by the MSR file, and spots can be manually flagged by user if desired. % Positive Controls Below Noise: 20 default(10) The percent of positive control probes that have signal below the local background noise level. This percentage tends to be higher when low concentration spikes fall below the threshold. A recommended setting is 30%, however, based on a large amount of arrays, we have made the setting at 20%. % of Negatives Above Noise: 5 default(0) The percent of negative control probes that have signals above local background. A recommended setting is 30%, however, based on a large amount of arrays, we have chosen 5% as our setting.
Description
doubly housed
Data processing
In preparation for normalization, probes were removed from the data sets if they were one of the negative or positive controls placed on the array by the manufacturer. Next, individual values were eliminated based on the quality flags assigned by the CodeLink Expression Analysis Software. Values were eliminated if they were flagged as M (spot was identified to be defective through image inspection at manufacturing), C (spot has a high level of background contamination), I (spot has an irregular shape), or S (spot has a high number of saturated pixels). Values were retained if they were flagged G (spot is good) or L (spot is below local background noise). In addition, to be able to take the log base 2 transformation of the background adjusted intensity values, all background adjusted intensity values below zero were replaced with the value 0.00001. The data was then normalized using a cyclic LOESS procedure executed in R with a procedure that accounted for the missing intensity values.
