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Summary
mRNA levels in cryopreserved BXD strain hepatotcytes (males at 60 days of age) immediately after thawing to 4 deg C. This is the "frozen state" mRNA status.
Experiment design
Control hepatocyte mRNA expression data prior to any treatment for young adult male BXD strains. This is esssentially the mRNA state of frozen hepatocytes.
About cases
| Sample ID | Varigenix Strain | Sex | RNA Concentrations ng/ul |
260/280 | 260/230 | Agilent Concentration(ng/ul) |
RIN |
| V1 | BXD32 | M | 1414.8 | 2.09 | 2.21 | 3,062 | 9.3 |
| V2 | BXD1 | M | 1608.9 | 2.11 | 2.24 | 3,102 | 9.3 |
| V3 | BXD5 | M | 1263.91 | 2.1 | 2.09 | 2,460 | 8.5 |
| V4 | BXD31 | M | 1793.95 | 2.1 | 2.22 | 3,387 | 8.5 |
| V5 | BXD8 | M | 1193.21 | 2.1 | 2.17 | 2,293 | 9.3 |
| V6 | BXD18 | M | 1041.05 | 2.13 | 2.17 | 2,115 | 9.2 |
| V7 | BXD42 | M | 398.28 | 1.99 | 2.28 | 1,519 | 8.3 |
| V8 | BXD29 | M | 477.37 | 2.02 | 2.24 | 1,301 | 8.1 |
| V9 | BXD21 | M | 414.6 | 2 | 2.27 | 1,174 | 8.3 |
| V10 | BXD27 | M | 438.61 | 2.02 | 2.27 | 1,100 | 8.1 |
| V11 | BXD16 | M | 436.21 | 2.01 | 2.27 | 868 | 8.5 |
| V12 | BXD19 | M | 489.12 | 2.01 | 2.26 | 1,277 | 8 |
| V13 | BXD22 | M | 1032.89 | 2.09 | 2.22 | 2,060 | 7.8 |
| V14 | BXD38 | M | 510.24 | 2.01 | 2.22 | 1,213 | 8 |
| V15 | BXD34 | M | 1021.26 | 2.09 | 2.24 | 1,514 | 8.2 |
| V16 | BXD20 | M | 330.85 | 2 | 2.27 | 901 | 8.1 |
| V17 | BXD15 | M | 1220.54 | 2.1 | 2.21 | 1,909 | 8.1 |
| V18 | BXD6 | M | 1001.04 | 2.09 | 2.2 | 1,523 | 8.3 |
| V19 | BXD13 | M | 1241.88 | 2.1 | 2.21 | 1,770 | 8.2 |
| V20 | BXD40 | M | 1032.04 | 2.08 | 2.22 | 1,985 | 8.2 |
| V21 | BXD14 | M | 881.62 | 2.11 | 2.19 | 481 | 8.7 |
| V22 | BXD2 | M | 1069.09 | 2.08 | 2.22 | 1,536 | 8.2 |
| V23 | BXD28 | M | 452.6 | 2 | 2.26 | 2,567 | 7.9 |
| V24 | BXD11 | M | 930.72 | 2.07 | 2.21 | 999 | 8.5 |
| V25 | BXD39 | M | 1177.27 | 2.07 | 2.23 | 838 | 8.7 |
About tissue
Hepatocytes prepared by Rob Kaiser at CRL Piedmont in Spring of 2011. Held in liquid nitrogen (vapor phase) until use Fall 2014 at UTHSC.
One vial of hepatocytes in 1 ml of freeze media was wiped with 70% ETOH, quick thawed by swift agitation in a 37 degree C water bath until a small amount of ice remained in the vial. Contents of the vial were removed with a sterile 1000 ul pipette tip and added to a 5 ml sterile polypropylene tube on ice containing 3 ml of ice-cold sterile 1x PBS. sitting in ice. One ml of sterile ice-cold 1x PBS was added to the original vial to remove any remaining cells and added to the 5 ml tube on ice. This process was quickly repeated with three more vials of cells. Four 5 ml tubes were centrifuged at 8,000 rpm at 4 degrees C for 4 minutes to pellet hepatocytes. PBS diluted freeze media was completely removed from the cell pellet. Entire process for sets of vials took about 7 minutes until RNA lysis buffer was added.
The QIAgen AllPrep DNA/RNA mini kit was used in conjunction with the QIAcube for purification of DNA and RNA from the hepatocytes. 600 ul of kit lysis buffer was added to pelleted cells, along with one 5 mm sterile stainless steel bead and cells were completely disrupted using the TissueLyser. The QIAcube protocol was used first for DNA extraction (held at -80 deg C for future use) and the flow-through containing total RNA was then used for the second purification.
Any residual DNA was removed from the RNA before Agilent quantification of concentration and RIN, using the QIAgen DNase I reagent, followed by ethanol precipitation and resuspension of DNA-free total RNA in 50 ul RNAse free water.
About platform
Affy Mouse Gene 1.0 ST (GPL6246)
About data processing
Standard 2z+8 of log2 data. RMA data were log2 transformed (adding an olffset of 1 to avoid negative values). Variance was stablized (i.e. each array was converted to a set of Z scores). Z scores were then multipled by 2. Mean Z was then shifted from 0 to to 8 units per array.
Contributors
Robert W. Williams, Roberrt E. Scott and colleagues. Cells were donated to Williams group by Varigenix Inc. and CRL Inc.
Citation
In progress.
Acknowledgment
We thank Varigenix and Charle River Laboratory for their donation of BXD cyropreserved hepatcytes. We thank Jesse Ingels for preparing RNA samples. We thank Lorne Rose and the UTHSC Molecular Resource Center for processing RNA samples and generating array data. We thank Arthur Centeno for data entry. We thank the UTHSC CITG and the UT-ORNL Governor's chair for support of microarray analysis.