C57B/6 strain, Age: 17 days, Tissue: Liver, Pooled RNA from Hdac3-null livers of 5 mice
Extracted molecule
total RNA
Extraction protocol
Tissue was kept in RNAlater overnight at 4 degrees, and homogenized in lysis buffer containing TCEP. Protcol from PerfectPure RNA Fibrous Tissue Kit (Mat # 2900304), was followed exactly, including DNase step, and RNA eluted in 40-60 microliters of nuclease free water, and stored at -80 degrees.
Label
DIG
Label protocol
All RNA Preps submitted to the VMSR were run on an Agilent 2100 Bioanalyzer to assess RNA integrity. Those samples meeting minimum requirements of RIN value of 7.0 and greater were used to generate targets for hybridization to ABI arrays. One (1) ug of Total RNA (30ng mRNA) was used to generate First Strand cDNA using the NanoAmp RT-IVT labeling kit according to manufacturer’s protocol (ABI, Cat#4365715). Following first strand synthesis, second strand synthesis was completed. The resulting cDNA was then purified using an ABI kit provided column and the entire reaction was used in an IVT reaction to generate DIG labeled cRNA. The cRNA was then purified using a kit provided column and assessed for quality on an Agilent Bioanalyzer. All reactions meeting ABI criteria in terms of quantity and size of target produced were fragmented and then hybridized to the appropriate array for 16 hours with agitation at 55C.
Hybridization protocol
All hybridization reagents, hybridization controls, wash reagents, and chemiluminescent reagents were provided in the ABI CL Detection Kit, part#4342142, and the manufacturer’s protocol was followed. Briefly, the arrays were equilibrated to room temperature and then pre-hybridized with a 1ml volume for 60’ with agitation (100RPM) at 55C per manufacturer’s protocol. During the pre-hybridization, the DIG-labelled targets were fragmented and then stored on ice until the pre-hyb was completed. Once pre-hyb was completed the targets were mixed with the appropriate reagents, including Hybridization controls, and the 0.5ml target was added to the pre-hybridization through the port on the array chamber. The arrays were immediately returned to the 55C Hybridization oven and agitated exactly 16 hours at 100RPM. The arrays were then washed and incubated with Anti-Dig-APAntibody for 20 minutes. Following antibody washes, the arrays were incubated with Chemiluminescence Enhancing Solution, washed a final time and then stored in the last wash buffer at room temperature. Substrate for the chemiluminescence reaction was added to each array individually and then the array was immediately imaged on the 1700 Chemiluminescent Analyzer.
Scan protocol
Following addition of the chemiluminescence reaction substrate, each array was immediately imaged on the 1700 Chemiluminescent Analyzer. Each imaging was completed in approximately 15 minutes and the images were assessed for QC/QA and a primary analysis was completed by the AB1700 Expression Array System Software (v 1.1.1).
Description
Hybridization controls were used per Manufacturer’s instruction (see Hybridization procedures).
Data processing
As described in the VMSR ABI user manual, the .csv file contains the t-test results on all probes that passed a 'detectability' filter. If a probe is not detected (S/N < 3) in both subgroups, it is not included in the t test. If a probe is not detected in more than 50% of samples in a subgroup, it is not detected in that subgroup. Data was run through the R script ABarray for quantile normalization. Log transformation was performed on quantile normalized data to generate the number in the VALUE column.
