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| Status |
Public on Jul 26, 2016 |
| Title |
BXD68 Male H1 13-Jul-05 |
| Sample type |
RNA |
| |
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| Source name |
BXD68_M_H1_13-Jul-05
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| Organism |
Mus musculus |
| Characteristics |
tissue: hippocampus
strain: BXD68
Sex: Male
age (days): 72
batch id: 2
pool size (n of cases used per rna sample): 2
rma outlier: 0.01
scale factor: 2.404
background average: 48.28
present: 0.521
absent: 0.459
marginal: 0.02
affx-b-actinmur (3'/5'): 1.3
affx-gapdhmur (3'/5'): 0.74
source: UTM RW
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| Treatment protocol |
BXD animals were obtained from UTHSC, UAB, or directly from The Jackson Laboratory (see Table 1 below). Animals were housed at UTHSC, Beth Israel Deaconess, or the Jackson Laboratory before sacrifice. Virtually all CXB animals were obtained directly at the Jackson Laboratory by Lu Lu. We thank Muriel Davisson for making it possible to collect these cases on site. Standard inbred strain stock was from The Jackson Laboratory, but most animals were housed or reared at UTHSC. Mice were euthanized and killed by cervical dislocation and brains were removed and placed in RNAlater prior to dissection. Cerebella and olfactory bulbs were removed; brains were hemisected, and both hippocampi were dissected whole by Hong Tao Zhang in the Lu lab. Hippocampal samples are very close to complete (see Lu et al., 2001) but probably include variable amounts of subiculum and fimbria. A great majority of animals used in this study were between 45 and 90 days of age (average of 66 days, maximum range from 41 to 196 days). All animals were sacrifice between 9 AM and 5 PM during the light phase.
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| Growth protocol |
Cells grown in mono layer are lysed directly in a culture dish by adding the RNA STAT-60TM (1 ml/3.5 cm petri dish) and passing the cell lysate several times through a pipette. Cells grown in suspension are sediment then lysed in the RNA STAT-60TM (1 ml per 5-10 x 106 cells) by repetitive pipetting. Washing calls before addition of the RNA STAT-60TM should be avoided as this increases the possibility of mRNA degradation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA Extraction: In brief, we used the RNA STAT-60 protocol (TEL-TEST B Bulletin No. 1), steps 5.1A (homogenization of tissue), 5.2 (RNA extraction), 5.3 (RNA precipitation), and 5.4 (RNA wash). In Step 5.4 we stopped after adding 75% ethanol (1 ml per 1 ml RNA STAT-60) and stored the mix at -80°C until further use. Before RNA labeling we thawed samples and proceeded with the remainder of Step 5.4; pelleting, drying, and redissolving the pellet in RNase-free water.
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| Label |
biotin
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| Label protocol |
The BXD genetic reference panel of recombinant inbred strains consists of just over 80 strains. The BXDs in this data set include 27 of the BXD strains made by Benjamin Taylor at the Jackson Laboratory in the 1970s and 1990s (BXD1 through BXD42). All of these strains are fully inbred, many well beyond the 100th filial (F) generation of inbreeding. We have also included 39 inbred (25 strains at F20+) and nearly inbred (14 strains between F14 and F20) BXD lines generated by Lu and Peirce. All of these strains, including those between F14 and F20, have been genotyped at 13,377 SNPs. Mouse Diversity Panel (MDP). We have profiled a MDP consisting 16 inbred strains and a pair of reciprocal F1 hybrids; B6D2F1 and D2B6F1.
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| |
|
| Hybridization protocol |
Samples were processed in the INIA Bioanalytical Core at the W. Harry Feinstone Center for Genomic Research, The University of Memphis, led by Thomas R. Sutter. All processing steps were performed by Shirlean Goodwin. In brief, RNA purity was evaluated using the 260/280 nm absorbance ratio, and values had to be greater than 1.8. The majority of samples were 1.9 to 2.1. RNA integrity was assessed using the Agilent Bioanalyzer 2100. We required an RNA integrity number (RIN) of greater than 8. This RIN value is based on the intensity ratio and amplitude of 18S and 28S rRNA signals. The standard Eberwine T7 polymerase method was used to catalyze the synthesis of cDNA template from polyA-tailed RNA using Superscript II reverse transcriptase (Invitrogen Inc.). The Enzo Life Sciences, Inc., BioArray High Yield RNA Transcript Labeling Kit (T7, Part No. 42655) was used to synthesize labeled cRNA. The cRNA was evaluated using both the 260/280 ratio (values of 2.0 or 2.1 are acceptable) and the Bioanalyzer output (a dark cRNA smear on the 2100 output centered roughly between 600 and 2000 nucleotides is required). Those samples that passed both QC steps (10% usually failed and new RNA samples had to be acquired and processed) were then sheared using a fragmentation buffer included in the Affymetrix GeneChip Sample Cleanup Module (Part No. 900371). Fragmented cRNA samples were either stored at -80°C until use or were immediately injected onto the array. The arrays were hybridized and washed following standard Affymetrix protocols.
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| Scan protocol |
All samples in a group were labeled on one day, except for a few cases that failed QC on their first pass. The hybridization station accommodates up to 20 samples, and for this reason each group was split into a large first set of 20 samples and a second set of 12 to 14 samples. Samples were washed in groups of four and then held in at 4°C until all 20 (or 12-14) arrays were ready to scan. The last four samples out of the wash stations were scanned directly. Samples were scanned in sets of four.
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| Data processing |
The expression values were generated using PDNN. The same simple steps described above were also applied to these values. Every microarray data set therefore has a mean expression of 8 with a standard deviation of 2. A 1 unit difference represents roughly a two-fold difference in expression level. Expression levels below 5 are usually close to background noise levels.
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| |
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| Submission date |
Jul 25, 2016 |
| Last update date |
Jul 26, 2016 |
| Contact name |
Robert W. Williams |
| Organization name |
University of Tennessee
|
| Department |
Department of Genetics, Genomics and Informatics
|
| Street address |
71 Manassas St.
|
| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38163 |
| Country |
USA |
| |
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| Platform ID |
GPL1261 |
| Series (1) |
| GSE84767 |
Genetics of the hippocampal transcriptome in mouse: a systematic survey and online neurogenomics resource |
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