title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta


title: Retina vs pool

status: Public on Aug 15 2008

type: RNA

city: Seattle

first: Amit

middle: V

last: Kulkarni

state: WA

address: 401 Terry Ave N

department: Genomics

country: USA

institute: Rosetta Inpharmatics / Merck Pharmaceuticals

description: Retina vs pool

hyb_protocol: Microarrays are incubated at 40°C for 48 hours in a rotating carousel.  Hybridizations to custom Agilent microarrays are completed as previously described (Hughes et al.Nat Biotech (2001), 19(4):342-7).  Microarrays are washed to remove non-specific hybridized sample. Afterwards, microarrays are dried in an ozone-free nitrogen chamber.

scan_protocol: Microarrays are scanned using the Agilent LP2 laser scanner. The scanner output is a Tiff file, which contains the quantitative hybridization data from each individual microarray. The Tiff files are then processed using Rosetta custom feature extraction software.

data_processing: Data were processed using the Rosetta Resolver® system. Rosetta's custom feature extraction software performs error modeling before data are loaded into the Resolver system. The Resolver system performs a squeeze operation that combines replicates of the same sequence in an array while applying error weighting. The error weighting consists of adjusting for additive and multiplicative noise. A P-value is generated and propagated throughout the system. The P-value represents the probability that a gene is expressed. The Resolver system allows users to set thresholds, below which genes of a P-value are considered to be significantly expressed. The Resolver system also combines multiple arrays using a squeezing process. If multiple spots reference one sequence, summarization is performed using an error-weighted average as described in Roland Stoughton and Hongyue Dai, Statistical Combining of Cell Expression Profiles. US Patent #6,351,712, February 26, 2002.

molecule: polyA RNA

organism: Homo sapiens

source_name: Retina

label_protocol: custom automated version of the aminoallyl MessageAmp II kit from Ambion.

extract_protocol: Total RNA isolated using Qiagen RNeasy spin columns with DNAse treatment; polyA+ RNA isolated from total RNA via magnetic bead-based mRNA extraction (Ambion, Poly(A) Purist)

tag: 48 diverse human tissues and cell lines were hybridized to a 17-array set.  Samples were purchased as pools from multiple donors, typically over 10 (Clontech, Mountainview, CA).

source_name: pool

tag: Pooled RNA from 20 diverse disease-free adult tissue pools comprised the reference pool.


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Brain, Cerebellum vs pool

description: Brain, Cerebellum vs pool

source_name: Brain, Cerebellum


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Thyroid vs pool

description: Thyroid vs pool

source_name: Thyroid


title: Ovary vs pool

description: Ovary vs pool

source_name: Ovary


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Skin vs pool

description: Skin vs pool

source_name: Skin


title: Bone Marrow vs pool

description: Bone Marrow vs pool

source_name: Bone Marrow


title: Colon vs pool

description: Colon vs pool

source_name: Colon


title: Skeletal Muscle vs pool

description: Skeletal Muscle vs pool

source_name: Skeletal Muscle


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe


title: Mammary gland vs pool

description: Mammary gland vs pool

source_name: Mammary gland


title: Adrenal Gland vs pool

description: Adrenal Gland vs pool

source_name: Adrenal Gland


title: Spinal Cord vs pool

description: Spinal Cord vs pool

source_name: Spinal Cord

source_name: pool


title: Adipose vs pool

description: Adipose vs pool

source_name: Adipose


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Stomach vs pool

description: Stomach vs pool

source_name: Stomach


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary


title: Jejunum vs pool

description: Jejunum vs pool

source_name: Jejunum


title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta


title: Thyroid vs pool

description: Thyroid vs pool

source_name: Thyroid


title: Lung vs pool

description: Lung vs pool

source_name: Lung


title: Kidney vs pool

description: Kidney vs pool

source_name: Kidney


title: Ileum vs pool

description: Ileum vs pool

source_name: Ileum


title: Retina vs pool

description: Retina vs pool

source_name: Retina


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Heart vs pool

description: Heart vs pool

source_name: Heart


title: Skin vs pool

description: Skin vs pool

source_name: Skin


title: Skeletal Muscle vs pool

description: Skeletal Muscle vs pool

source_name: Skeletal Muscle


title: Mammary gland vs pool

description: Mammary gland vs pool

source_name: Mammary gland

source_name: pool


title: Fetal Lung vs pool

description: Fetal Lung vs pool

source_name: Fetal Lung


title: Ovary vs pool

description: Ovary vs pool

source_name: Ovary


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary


title: Fetal Brain vs pool

description: Fetal Brain vs pool

source_name: Fetal Brain


title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Fetal Liver vs pool

description: Fetal Liver vs pool

source_name: Fetal Liver


title: Stomach vs pool

description: Stomach vs pool

source_name: Stomach


title: Ileum vs pool

description: Ileum vs pool

source_name: Ileum


title: Jejunum vs pool

description: Jejunum vs pool

source_name: Jejunum


title: Colon vs pool

description: Colon vs pool

source_name: Colon


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Lung vs pool

description: Lung vs pool

source_name: Lung


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Brain, Cerebellum vs pool

description: Brain, Cerebellum vs pool

source_name: Brain, Cerebellum

source_name: pool


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Bone Marrow vs pool

description: Bone Marrow vs pool

source_name: Bone Marrow


title: Heart vs pool

description: Heart vs pool

source_name: Heart


title: Kidney vs pool

description: Kidney vs pool

source_name: Kidney


title: Fetal Brain vs pool

description: Fetal Brain vs pool

source_name: Fetal Brain


title: Thyroid vs pool

description: Thyroid vs pool

source_name: Thyroid


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Adipose vs pool

description: Adipose vs pool

source_name: Adipose


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Adrenal Gland vs pool

description: Adrenal Gland vs pool

source_name: Adrenal Gland


title: Spinal Cord vs pool

description: Spinal Cord vs pool

source_name: Spinal Cord


title: Colon vs pool

description: Colon vs pool

source_name: Colon


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Fetal Lung vs pool

description: Fetal Lung vs pool

source_name: Fetal Lung


title: Stomach vs pool

description: Stomach vs pool

source_name: Stomach


title: Skin vs pool

description: Skin vs pool

source_name: Skin


title: Fetal Liver vs pool

description: Fetal Liver vs pool

source_name: Fetal Liver

source_name: pool


title: Skeletal Muscle vs pool

description: Skeletal Muscle vs pool

source_name: Skeletal Muscle


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Retina vs pool

description: Retina vs pool

source_name: Retina


title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Brain, Cerebellum vs pool

description: Brain, Cerebellum vs pool

source_name: Brain, Cerebellum


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary


title: Adrenal Gland vs pool

description: Adrenal Gland vs pool

source_name: Adrenal Gland


title: Bone Marrow vs pool

description: Bone Marrow vs pool

source_name: Bone Marrow


title: Adipose vs pool

description: Adipose vs pool

source_name: Adipose


title: Ovary vs pool

description: Ovary vs pool

source_name: Ovary


title: Thyroid vs pool

description: Thyroid vs pool

source_name: Thyroid


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Lung vs pool

description: Lung vs pool

source_name: Lung


title: Stomach vs pool

description: Stomach vs pool

source_name: Stomach

source_name: pool


title: Skin vs pool

description: Skin vs pool

source_name: Skin


title: Mammary gland vs pool

description: Mammary gland vs pool

source_name: Mammary gland


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Kidney vs pool

description: Kidney vs pool

source_name: Kidney


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe


title: Jejunum vs pool

description: Jejunum vs pool

source_name: Jejunum


title: Ileum vs pool

description: Ileum vs pool

source_name: Ileum


title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta


title: Spinal Cord vs pool

description: Spinal Cord vs pool

source_name: Spinal Cord


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary


title: Heart vs pool

description: Heart vs pool

source_name: Heart


title: Fetal Lung vs pool

description: Fetal Lung vs pool

source_name: Fetal Lung


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Fetal Brain vs pool

description: Fetal Brain vs pool

source_name: Fetal Brain


title: Fetal Liver vs pool

description: Fetal Liver vs pool

source_name: Fetal Liver


title: Skeletal Muscle vs pool

description: Skeletal Muscle vs pool

source_name: Skeletal Muscle

source_name: pool


title: Lung vs pool

description: Lung vs pool

source_name: Lung


title: Colon vs pool

description: Colon vs pool

source_name: Colon


title: Retina vs pool

description: Retina vs pool

source_name: Retina


title: Ovary vs pool

description: Ovary vs pool

source_name: Ovary


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Mammary gland vs pool

description: Mammary gland vs pool

source_name: Mammary gland


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe


title: Jejunum vs pool

description: Jejunum vs pool

source_name: Jejunum


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Bone Marrow vs pool

description: Bone Marrow vs pool

source_name: Bone Marrow


title: Heart vs pool

description: Heart vs pool

source_name: Heart


title: Ileum vs pool

description: Ileum vs pool

source_name: Ileum


title: Brain, Cerebellum vs pool

description: Brain, Cerebellum vs pool

source_name: Brain, Cerebellum


title: Adrenal Gland vs pool

description: Adrenal Gland vs pool

source_name: Adrenal Gland


title: Fetal Brain vs pool

description: Fetal Brain vs pool

source_name: Fetal Brain


title: Adipose vs pool

description: Adipose vs pool

source_name: Adipose

source_name: pool


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Thyroid vs pool

description: Thyroid vs pool

source_name: Thyroid


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Stomach vs pool

description: Stomach vs pool

source_name: Stomach


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Spinal Cord vs pool

description: Spinal Cord vs pool

source_name: Spinal Cord


title: Fetal Lung vs pool

description: Fetal Lung vs pool

source_name: Fetal Lung


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Retina vs pool

description: Retina vs pool

source_name: Retina


title: Skin vs pool

description: Skin vs pool

source_name: Skin


title: Colon vs pool

description: Colon vs pool

source_name: Colon


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe


title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta


title: Skeletal Muscle vs pool

description: Skeletal Muscle vs pool

source_name: Skeletal Muscle


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Fetal Liver vs pool

description: Fetal Liver vs pool

source_name: Fetal Liver


title: Brain, Cerebellum vs pool

description: Brain, Cerebellum vs pool

source_name: Brain, Cerebellum

source_name: pool


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary


title: Adrenal Gland vs pool

description: Adrenal Gland vs pool

source_name: Adrenal Gland


title: Thymus vs pool

description: Thymus vs pool

source_name: Thymus


title: Ovary vs pool

description: Ovary vs pool

source_name: Ovary


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Stomach vs pool

description: Stomach vs pool

source_name: Stomach


title: Bone Marrow vs pool

description: Bone Marrow vs pool

source_name: Bone Marrow


title: Mammary gland vs pool

description: Mammary gland vs pool

source_name: Mammary gland


title: Spinal Cord vs pool

description: Spinal Cord vs pool

source_name: Spinal Cord


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Lung vs pool

description: Lung vs pool

source_name: Lung


title: Retina vs pool

description: Retina vs pool

source_name: Retina


title: Kidney vs pool

description: Kidney vs pool

source_name: Kidney


title: Thyroid vs pool

description: Thyroid vs pool

source_name: Thyroid


title: Adipose vs pool

description: Adipose vs pool

source_name: Adipose


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta

source_name: pool


title: Ileum vs pool

description: Ileum vs pool

source_name: Ileum


title: Heart vs pool

description: Heart vs pool

source_name: Heart


title: Skin vs pool

description: Skin vs pool

source_name: Skin


title: Skeletal Muscle vs pool

description: Skeletal Muscle vs pool

source_name: Skeletal Muscle


title: Jejunum vs pool

description: Jejunum vs pool

source_name: Jejunum


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Ovary vs pool

description: Ovary vs pool

source_name: Ovary


title: Fetal Lung vs pool

description: Fetal Lung vs pool

source_name: Fetal Lung


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Fetal Liver vs pool

description: Fetal Liver vs pool

source_name: Fetal Liver


title: Lung vs pool

description: Lung vs pool

source_name: Lung


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Mammary gland vs pool

description: Mammary gland vs pool

source_name: Mammary gland


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Fetal Brain vs pool

description: Fetal Brain vs pool

source_name: Fetal Brain


title: Colon vs pool

description: Colon vs pool

source_name: Colon

source_name: pool


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe


title: Brain, Cerebellum vs pool

description: Brain, Cerebellum vs pool

source_name: Brain, Cerebellum


title: Kidney vs pool

description: Kidney vs pool

source_name: Kidney


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Heart vs pool

description: Heart vs pool

source_name: Heart


title: Bone Marrow vs pool

description: Bone Marrow vs pool

source_name: Bone Marrow


title: Ileum vs pool

description: Ileum vs pool

source_name: Ileum


title: Adrenal Gland vs pool

description: Adrenal Gland vs pool

source_name: Adrenal Gland


title: SLN:1480 - A - mAdbID:38184

status: Public on Jun 03 2009

city: Bethesda

first: Louis

middle: M.

last: Staudt

state: MD

address: 9000 Rockville Pike, Bldg 10, Rm 4N114

department: Lymphoid Malignancies Branch

institute: National Cancer Institute

description: mAdb experiment ID: 38184

hyb_protocol: Affymetrix Hybridization Protocol Other: Following fragmentation, 20 micrograms of cRNA were hybridized for 16 hours at 45C on U133A/B GeneChips. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: Affy Scanning Protocol Other: Scanning was performed by the Affymetrix 3000 Scanner.

data_processing:
MAS 5.0 Data Processing Protocol
Calculation Method: The data were analyzed with Microarray Suite version 5.0 (MA S 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

label: biotin

label_protocol:
Affymetrix Labeling Protocol
Other: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 1 microg mRNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol:
FastTrack 2.0 mRNA Isolation Kit
Extraction method: oligo dt cellulose
Other: mRNA was extracted using the FastTrack 2.0 mRNA Isolation Kit according to manufacturer's instructions (Invitrogen, Carlsbad, CA).

tag: tissue

value: Lymph node

tag: disease state

value: Follicular Lymphoma

tag: individual

tag: clinical info

value: Follow up status:alive

value: Follow up years:16.29

value: Stage >2:no

value: LDH ratio >1:yes

value: Number of extranodal sites >1:no

value: Age >60:no

value: IPI score:1

value: Immune response 1:9.4

value: Immune response 2:8.67

value: With translocation:no


title: Adipose vs pool

description: Adipose vs pool

source_name: Adipose


title: Spinal Cord vs pool

description: Spinal Cord vs pool

source_name: Spinal Cord


title: Fetal Lung vs pool

description: Fetal Lung vs pool

source_name: Fetal Lung


title: Stomach vs pool

description: Stomach vs pool

source_name: Stomach


title: Thyroid vs pool

description: Thyroid vs pool

source_name: Thyroid


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Colon vs pool

description: Colon vs pool

source_name: Colon

source_name: pool


title: Skeletal Muscle vs pool

description: Skeletal Muscle vs pool

source_name: Skeletal Muscle


title: Skin vs pool

description: Skin vs pool

source_name: Skin


title: Thymus vs pool

description: Thymus vs pool

source_name: Thymus


title: Fetal Liver vs pool

description: Fetal Liver vs pool

source_name: Fetal Liver


title: Fetal Brain vs pool

description: Fetal Brain vs pool

source_name: Fetal Brain


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Retina vs pool

description: Retina vs pool

source_name: Retina


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe


title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Bone Marrow vs pool

description: Bone Marrow vs pool

source_name: Bone Marrow


title: Ovary vs pool

description: Ovary vs pool

source_name: Ovary


title: Adrenal Gland vs pool

description: Adrenal Gland vs pool

source_name: Adrenal Gland


title: Thyroid vs pool

description: Thyroid vs pool

source_name: Thyroid


title: Brain, Cerebellum vs pool

description: Brain, Cerebellum vs pool

source_name: Brain, Cerebellum


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary


title: Mammary gland vs pool

description: Mammary gland vs pool

source_name: Mammary gland

source_name: pool


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Testis vs pool

description: Testis vs pool

source_name: Testis


title: Adipose vs pool

description: Adipose vs pool

source_name: Adipose


title: Spinal Cord vs pool

description: Spinal Cord vs pool

source_name: Spinal Cord


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Lung vs pool

description: Lung vs pool

source_name: Lung


title: Skin vs pool

description: Skin vs pool

source_name: Skin


title: Kidney vs pool

description: Kidney vs pool

source_name: Kidney


title: Skeletal Muscle vs pool

description: Skeletal Muscle vs pool

source_name: Skeletal Muscle


title: Jejunum vs pool

description: Jejunum vs pool

source_name: Jejunum


title: Ileum vs pool

description: Ileum vs pool

source_name: Ileum


title: Retina vs pool

description: Retina vs pool

source_name: Retina


title: Placenta vs pool

description: Placenta vs pool

source_name: Placenta


title: Thymus vs pool

description: Thymus vs pool

source_name: Thymus


title: Heart vs pool

description: Heart vs pool

source_name: Heart


title: Stomach vs pool

description: Stomach vs pool

source_name: Stomach


title: Pituitary vs pool

description: Pituitary vs pool

source_name: Pituitary

source_name: pool


title: Fetal Lung vs pool

description: Fetal Lung vs pool

source_name: Fetal Lung


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Fetal Brain vs pool

description: Fetal Brain vs pool

source_name: Fetal Brain


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Mammary gland vs pool

description: Mammary gland vs pool

source_name: Mammary gland


title: Colon vs pool

description: Colon vs pool

source_name: Colon


title: Fetal Liver vs pool

description: Fetal Liver vs pool

source_name: Fetal Liver


title: Lung vs pool

description: Lung vs pool

source_name: Lung


title: Ovary vs pool

description: Ovary vs pool

source_name: Ovary


title: Jejunum vs pool

description: Jejunum vs pool

source_name: Jejunum


title: Liver vs pool

description: Liver vs pool

source_name: Liver


title: Brain, Thalamus vs pool

description: Brain, Thalamus vs pool

source_name: Brain, Thalamus


title: Kidney vs pool

description: Kidney vs pool

source_name: Kidney


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Brain, Cerebellum vs pool

description: Brain, Cerebellum vs pool

source_name: Brain, Cerebellum


title: Ileum vs pool

description: Ileum vs pool

source_name: Ileum


title: Brain, Frontal Lobe vs pool

description: Brain, Frontal Lobe vs pool

source_name: Brain, Frontal Lobe

source_name: pool


title: Bone Marrow vs pool

description: Bone Marrow vs pool

source_name: Bone Marrow


title: Heart vs pool

description: Heart vs pool

source_name: Heart


title: Adrenal Gland vs pool

description: Adrenal Gland vs pool

source_name: Adrenal Gland


title: Prostate vs pool

description: Prostate vs pool

source_name: Prostate


title: Fetal Lung vs pool

description: Fetal Lung vs pool

source_name: Fetal Lung


title: Colon vs pool

description: Colon vs pool

source_name: Colon


title: Fetal Liver vs pool

description: Fetal Liver vs pool

source_name: Fetal Liver


title: Adipose vs pool

description: Adipose vs pool

source_name: Adipose


title: Fetal Kidney vs pool

description: Fetal Kidney vs pool

source_name: Fetal Kidney


title: Fetal Brain vs pool

description: Fetal Brain vs pool

source_name: Fetal Brain


title: Joosse 2007: FFPE tumor #6 - manual hybridization

status: Public on Feb 23 2007

type: genomic

city: Hamburg

first: Simon

middle: A

last: Joosse

address: Martinistrasse 52

department: Institute of Tumor Biology

country: Germany

institute: University Medical Center Hamburg-Eppendorf

hyb_protocol: Step 1: Wash at 37°C, 2x SSC (pH 7), 1 wash run for 30 sec, no soak Step 2: Probe injection (pre-hyb mix) at 37°C Step 3: Hybridisation at 37°C for 01:00:00, Agitation Frequency: high Step 4: Wash at 37°C, 2x SSC (pH 7), 1 run, wash time 15 sec, no soak Step 5: Probe injection (sample) at 37°C Step 6: Hybridisation at 37°C for 69:00:00, Agitation Frequency: high Step 7: Wash at 37°C, 2x SSC, 0.1% SDS, 12 runs, wash time 1 min, soak time 1 min Step 8: Wash at 68°C, 2x SSC, 0.1% SDS, 6 runs, wash time 1 min, soak time 1 min Step 9: Wash at 68°C, 2x SSC (pH 7), 2 runs, wash time 1:30 min, soak time 1 min Step 10: Wash at 23°C, 0.1x SSC, 1 wash run for 1:30 sec, no soak Step 11: Slide drying step at 30°C for 2 min, final manifold cleaning using N2 gas

scan_protocol: Slides were scanned with an Agilent DNA Microarray Scanner BA on the same day.

data_processing: Data processing included signal intensity measurement in ImaGene Software and median pintip (c.q. subarray) normalization.

molecule: genomic DNA

source_name: Formalin-fixed, paraffin-embedded tumor #6

label_protocol: According to the manufacturers’ instructions (Kreatech Biotechnology, Amsterdam, http://www.kreatech.com/).

extract_protocol: DNA is extracted as described in: Van Beers EH, Joosse SA, Ligtenberg MJ, Fles R, Hogervorst FBL, Verhoef S, Nederlof PM: A multiplex PCR predictor for aCGH success of FFPE samples. Br J Cancer. 2006 Jan;94(2):333-7

tag: organ

value: mammary gland; breast

tag: disease

value: infiltrating ductal carcinoma

source_name: Women reference pool

extract_protocol:
Equipment and reagents:
Lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA), DNAzol (Invitrogen), TE buffer pH 8.0, 10 mM Tris.HCl, 1 mM EDTA;
Lysis:
1. Mix the blood carefully and decant in a 50 ml tube;
2. Rinse the EDTA tube with lysis buffer and decant this together with the blood;
3. Fill the tube to 50 ml with lysis buffer and mix;
4. Put the tube on ice for 10 minutes;
5. Centrifuge at 3000 rpm for 10 minutes at 4°C;
6. Poor off the supernatant;
7. Carefully resuspent cell pellet in 50 ml lysis buffer;
8. Centrifuge at 3000 rpm for 10 min at 4°C;
9. Poor off supernatant;
10. Repeat wash steps until the supernatant is a clear solution;
11. Sample can be stored in liquid nitrogen or at -70°C;
Genomic DNA isolation:
1. Add 1 ml DNAzol to the pellet and mix by pipeting until clear solution is left;
2. Add 500 ml 100% EtOH and mix carefully;
3. Transfer the DNA pellet with a sterile öse to tube with 1 ml 70% EtOH;
4. Wash the DNA and press as many EtOH out of the pellet as possible;
5. Transfer the DNA to a 2 ml Eppendorf tube with 1 ml TE buffer;
6. Solute by rotating overnight at 37°C;

tag: A pool of DNA from peripheral blood lymphocytes, from 7 apperently healty women.


title: Joosse 2007: FFPE tumor #5 - automated hybridization

source_name: Formalin-fixed, paraffin-embedded tumor #5


title: Joosse 2007: FFPE tumor #6 - automated hybridization


title: Joosse 2007: FFPE tumor #5 - manual hybridization


title: Brain, Hippocampus vs pool

description: Brain, Hippocampus vs pool

source_name: Brain, Hippocampus


title: Joosse 2007: FFPE tumor #4 - automated hybridization

source_name: Formalin-fixed, paraffin-embedded tumor #4


title: Joosse 2007: SKBR3 - hybridized at 45C for 72 hours

source_name: SKBR3 cell-line

extract_protocol: Cells were suspended in 1 ml DNAzol reagent, centrifuged at 14000rpm for 10 min at 4C. The supernatant was transferred to a new tube and 0.5 ethanol absolute was added to precipitate the DNA with swirling. The DNA precipitate was transferred to a new tube and washed with 1 ml 75% ethanol. The ethanol was removed and the DNA was disolved in 50ul TE buffer.

value: adenocarcinoma

tag: derived from metastatic site

value: pleural effusion

tag: age

value: 43 years

tag: gender

value: female

tag: ethnicity

value: Caucasian

extract_protocol:
Equipment and reagents
Lysis buffer (155 mM NH4Cl,	10 mM KHCO3, 1 mM EDTA),	DNAzol (Invitrogen), TE buffer pH 8.0, 10 mM Tris.HCl, 1 mM EDTA
Lysis
1.	Mix the blood carefully and decant in a 50 ml tube
2.	Rinse the EDTA tube with lysis buffer and decant this together with the blood
3.	Fill the tube to 50 ml with lysis buffer and mix
4.	Put the tube on ice for 10 minutes
5.	Centrifuge at 3000 rpm for 10 minutes at 4°C
6.	Poor off the supernatant
7.	Carefully resuspent cell pellet in 50 ml lysis buffer
8.	Centrifuge at 3000 rpm for 10 min at 4°C
9.	Poor off supernatant
10.	Repeat wash steps until the supernatant is a clear solution
11.	Sample can be stored in liquid nitrogen or at -70°C
Genomic DNA isolation
1.	Add 1 ml DNAzol to the pellet and mix by pipeting until clear solution is left
2.	Add 500 ml 100% EtOH and mix carefully
3.	Transfer the DNA pellet with a sterile öse to tube with 1 ml 70% EtOH
4.	Wash the DNA and press as many EtOH out of the pellet as possible
5.	Transfer the DNA to a 2 ml Eppendorf tube with 1 ml TE buffer
6.	Solute by rotating overnight at 37°C


title: Joosse 2007: FFPE tumor #4 - manual hybridization


title: Joosse 2007: SKBR3 - hybridized at 37C for 24 hours


title: Joosse 2007: FFPE tumor #3 - manual hybridization

source_name: Formalin-fixed, paraffin-embedded tumor #3


title: Joosse 2007: FFPE tumor #3 - automated hybridization


title: Joosse 2007: SKBR3 - automated hybridization - experiment 2


title: Joosse 2007: FFPE SKBR3 - automated hybridization


title: Joosse 2007: SKBR3 - manual hybridization

hyb_protocol: Hybridization as described in: Van Beers EH, Joosse SA, Ligtenberg MJ, Fles R, Hogervorst FBL, Verhoef S, Nederlof PM: A multiplex PCR predictor for aCGH success of FFPE samples. Br J Cancer. 2006 Jan;94(2):333-7

extract_protocol:
Equipment and reagents
Lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA), DNAzol (Invitrogen), TE buffer pH 8.0, 10 mM Tris.HCl, 1 mM EDTA
Lysis
1. Mix the blood carefully and decant in a 50 ml tube
2. Rinse the EDTA tube with lysis buffer and decant this together with the blood
3. Fill the tube to 50 ml with lysis buffer and mix
4. Put the tube on ice for 10 minutes
5. Centrifuge at 3000 rpm for 10 minutes at 4°C
6. Poor off the supernatant
7. Carefully resuspent cell pellet in 50 ml lysis buffer
8. Centrifuge at 3000 rpm for 10 min at 4°C
9. Poor off supernatant
10. Repeat wash steps until the supernatant is a clear solution
11. Sample can be stored in liquid nitrogen or at -70°C
Genomic DNA isolation
1. Add 1 ml DNAzol to the pellet and mix by pipeting until clear solution is left
2. Add 500 ml 100% EtOH and mix carefully
3. Transfer the DNA pellet with a sterile öse to tube with 1 ml 70% EtOH
4. Wash the DNA and press as many EtOH out of the pellet as possible
5. Transfer the DNA to a 2 ml Eppendorf tube with 1 ml TE buffer
6. Solute by rotating overnight at 37°C


title: Joosse 2007: SKBR3 - hybridized at 42C for 72 hours


title: Adipose vs pool

description: Adipose vs pool

source_name: pool


title: Joosse 2007: Tumor 2 - hybridization buffer at pH 8 - experiment 1

source_name: Formalin-fixed, paraffin-embedded tumor 2


title: Joosse 2007: SKBR3 - hybridized at 37C for 72 hours


title: Joosse 2007: SKBR3 - automated hybridization - experiment 1

extract_protocol: Cells weere suspended in 1 ml DNAzol reagent, centrifuged at 14000rpm for 10 min at 4C. The supernatant was transferred to a new tube and 0.5 ethanol absolute was added to precipitate the DNA with swirling. The DNA precipitate was transferred to a new tube and washed with 1 ml 75% ethanol. The ethanol was removed and the DNA was disolved in 50ul TE buffer.


title: Joosse 2007: Tumor 2 - hybridization buffer at pH 8 - experiment 2


title: Joosse 2007: Tumor 2 - hybridization buffer at pH 7 - experiment 2


title: Joosse 2007: Tumor 2 - hybridization buffer at pH 7 - experiment 1


title: Joosse 2007: Tumor 2 - hybridization buffer at pH 6 - experiment 1


title: Fetal Lung vs pool

description: Fetal Lung vs pool


title: Joosse 2007: SKBR3 CGH profile - 15% 50kDa dextran sulphate

extract_protocol: Cells were suspended in 1 ml DNAzol reagent, centrifuged at 14000rpm for 10 min at 4C. The supernatant was transferred to a new tube and 0.5 ethanol absolute was added to precipitate the DNA with swirling. The DNA precipitate was transferred to a new tube and washed with 1 ml 75% ethanol. The ethanol was removed and the DNA was disolved in 50ul TE buffer

extract_protocol:
Equipment and reagents:
Lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA), DNAzol (Invitrogen), TE buffer pH 8.0, 10 mM Tris.HCl, 1 mM EDTA;
Lysis:
1. Mix the blood carefully and decant in a 50 ml tube;
2. Rinse the EDTA tube with lysis buffer and decant this together with the blood;
3. Fill the tube to 50 ml with lysis buffer and mix;
4. Put the tube on ice for 10 minutes;
5. Centrifuge at 3000 rpm for 10 minutes at 4°C;
6. Poor off the supernatant;
7. Carefully resuspent cell pellet in 50 ml lysis buffer;
8. Centrifuge at 3000 rpm for 10 min at 4°C;
9. Poor off supernatant;
10. Repeat wash steps until the supernatant is a clear solution;
11. Sample can be stored in liquid nitrogen or at -70°C;
Genomic DNA isolation:
1. Add 1 ml DNAzol to the pellet and mix by pipeting until clear solution is left;
2. Add 500 ml 100% EtOH and mix carefully;
3. Transfer the DNA pellet with a sterile öse to tube with 1 ml 70% EtOH;
4. Wash the DNA and press as many EtOH out of the pellet as possible;
5. Transfer the DNA to a 2 ml Eppendorf tube with 1 ml TE buffer;
6. Solute by rotating overnight at 37°C


title: Joosse 2007: SKBR3 CGH profile - 20% 50kDa dextran sulphate


title: Joosse 2007: SKBR3 CGH profile - 20% 5kDa dextran sulphate


title: Joosse 2007: SKBR3 CGH profile - 17.5% 10kDa dextran sulphate


title: Joosse 2007: SKBR3 CGH profile - 20% 10kDa dextran sulphate


title: Joosse 2007: Tumor 2 - hybridization buffer at pH 6 - experiment 2


title: Joosse 2007: Tumor 2 - post-hybridization wash at 65°C - 2


title: Joosse 2007: SKBR3 CGH profile - 15% 10kDa dextran sulphate


title: Joosse 2007: SKBR3 CGH profile - 17.5% 5kDa dextran sulphate


title: Joosse 2007: SKBR3 CGH profile - 17.5% 50kDa dextran sulphate


title: Joosse 2007: Tumor 2 - post-hybridization wash at 68°C - 2

value: inviltrating ductal carcinoma


title: Joosse 2007: SKBR3 CGH profile - 15% dextran sulphate, hybridization temperature of 37°C


title: Joosse 2007: SKBR3 CGH profile - 15% dextran sulphate, hybridization temperature of 42°C


title: Pool Ts1Cje_P15_hyb1

status: Public on Dec 05 2004

city: Paris

first: Luce

last: Dauphinot

address: 47 boulevard de l'hôpital

department: ICM

country: France

institute: INSERM U1127-CNRS UMR7225-UPMC

description:
Pool of total RNA from 3 Ts1Cje cerebella (segmental trisomy of chromosome 16) at P15 postnatal stage
Keywords = Down syndrome
Keywords = Chromosome 21
Keywords = Transcriptome
Keywords = Microarray
Keywords = Brain
Keywords = Cerebellum
Keywords = Development

hyb_protocol: Biotinylated cRNA were hybridized to Affymetrix Murine Genome U74A version 2 microarrays (Affymetrix, Inc., Santa Clara, CA, USA) on the Curie Institute Affymetrix microarray platform (Paris, France), according to the Affymetrix procedures. Each pool was hybridized in duplicate on independent U74Av2 microarrays.

scan_protocol: According to standard Affymetrix procedures.

data_processing: After hybridization, signal intensities were calculated using the Affymetrix GeneChip® software MAS 5.0

molecule: total RNA

organism: Mus musculus

source_name: Pool Ts1Cje_P15

label_protocol:
An aliquot 20µg of total RNA from each pool was
converted to cDNA and then to biotinylated cRNA according to the Affymetrix procedures.

extract_protocol:
Total RNA was extracted from frozen individual cerebellum
and treated with DNase using RNeasy Midi kit (Qiagen).
The quality of each RNA sample was then checked using
the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.,
Palo Alto, CA, USA).

tag: ploidy

value: Ts1Cje (segmental trisomy of chromosome 16)

value: cerebellum

tag: developmental stage

value: P15 postnatal stage


title: Joosse 2007: SKBR3 CGH profile - 10% dextran sulphate, hybridization temperature of 42°C


title: Joosse 2007: Tumor 2 - post-hybridization wash at 65°C - 1


title: Pool Ts1Cje_P30_hyb2

description:
Pool of total RNA from 3 Ts1Cje cerebella (segmental trisomy of chromosome 16) at P30 postnatal stage
Keywords = Down syndrome
Keywords = Chromosome 21
Keywords = Transcriptome
Keywords = Microarray
Keywords = Brain
Keywords = Cerebellum
Keywords = Development

source_name: Pool Ts1Cje_P30

value: P30 postnatal stage


title: Joosse 2007: SKBR3 CGH profile - 15% 5kDa dextran sulphate


title: Joosse 2007: SKBR3 CGH profile - 10% dextran sulphate, hybridization temperature of 37°C


title: Pool Ts1Cje_P30_hyb1


title: Pool Eu_P30_hyb2

description:
Pool of total RNA from 2 euploid cerebella at P30 postnatal stage
Keywords = Down syndrome
Keywords = Chromosome 21
Keywords = Transcriptome
Keywords = Microarray
Keywords = Brain
Keywords = Cerebellum
Keywords = Development

source_name: Pool Eu_P30

value: euploid


title: Pool Ts1Cje_P0_hyb1

description:
Pool of total RNA from 3 Ts1Cje cerebella (segmental trisomy of chromosome 16) at P0 postnatal stage
Keywords = Down syndrome
Keywords = Chromosome 21
Keywords = Transcriptome
Keywords = Microarray
Keywords = Brain
Keywords = Cerebellum
Keywords = Development

source_name: Pool Ts1Cje_P0

value: P0 postnatal stage


title: Pool Eu_P30_hyb1


title: Pool Eu_P0_hyb2

description:
Pool of total RNA from 3 euploid cerebella at P0 postnatal stage
Keywords = Down syndrome
Keywords = Chromosome 21
Keywords = Transcriptome
Keywords = Microarray
Keywords = Brain
Keywords = Cerebellum
Keywords = Development

source_name: Pool Eu_P0


title: Pool Eu_P15_hyb1

description:
Pool of total RNA from 3 euploid cerebella at P15 postnatal stage
Keywords = Down syndrome
Keywords = Chromosome 21
Keywords = Transcriptome
Keywords = Microarray
Keywords = Brain
Keywords = Cerebellum
Keywords = Development

source_name: Pool Eu_P15


title: Joosse 2007: Tumor 2 - post-hybridization wash at 68°C - 1


title: Pool Eu_P0_hyb1


title: Pool Ts1Cje_P15_hyb2


title: Pool Eu_P15_hyb2


title: Pool Ts1Cje_P0_hyb2


title: MLD_Day0_Oil_Ut1

status: Public on Oct 28 2004

city: Okazaki

first: Atsuko

last: Suzuki

state: Aichi

address: Myodaiji, Higashiyama 5-1

department: Center for Integrative Bioscience

country: Japan

institute: National Institutes of Natural Sciences

description:
Pregnant mice, ICR (SLC, Tokyo, Japan), were given daily oil injections from fetal day 10 to 18. Total RNA was extracted at day of birth from three litters. One µg of total RNA were used to synthesize cDNA, which was then used to generate biotinylated cRNA
Keywords = uterus
Keywords = newborn

source_name: Murine uterus


title: MLD_Day0_Oil_Vg3

description:
Pregnant mice, ICR (SLC, Tokyo, Japan), were given daily oil injections from fetal day 10 to 18. Total RNA was extracted at day of birth from three litters. One µg of total RNA were used to synthesize cDNA, which was then used to generate biotinylated cRNA
Keywords = vagina
Keywords = newborn

source_name: Murine vagina


title: MLD_Day0_Oil_Ovi3

description:
Pregnant mice, ICR (SLC, Tokyo, Japan), were given daily oil injections from fetal day 10 to 18. Total RNA was extracted at day of birth from three litters. One µg of total RNA were used to synthesize cDNA, which was then used to generate biotinylated cRNA
Keywords = oviduct
Keywords = newborn

source_name: Murine oviduct


title: MLD_Day0_DES_Vg2

description:
Pregnant mice, ICR (SLC, Tokyo, Japan), were given daily injections of 67 µg DES /kg maternal body weight from fetal day 10 to 18. Total RNA was extracted at day of birth from three litters. One µg of total RNA were used to synthesize cDNA, which was then used to generate biotinylated cRNA
Keywords = vagina
Keywords = newborn
Keywords = DES-treatment


title: MLD_Day0_Oil_Vg2


title: MLD_Day0_Oil_Vg1


title: MLD_Day0_DES_Ut3

description:
Pregnant mice, ICR (SLC, Tokyo, Japan), were given daily injections of 67 µg DES /kg maternal body weight from fetal day 10 to 18. Total RNA was extracted at day of birth from three litters. One µg of total RNA were used to synthesize cDNA, which was then used to generate biotinylated cRNA
Keywords = uterus
Keywords = newborn
Keywords = DES-treatment


title: MLD_Day0_Oil_Ut2


title: MLD_Day0_DES_Ovi3

description:
Pregnant mice, ICR (SLC, Tokyo, Japan), were given daily injections of 67 µg DES /kg maternal body weight from fetal day 10 to 18. Total RNA was extracted at day of birth from three litters. One µg of total RNA were used to synthesize cDNA, which was then used to generate biotinylated cRNA
Keywords = oviduct
Keywords = newborn
Keywords = DES-treatment


title: MLD_Day0_DES_Ut2


title: MLD_Day0_Oil_Ovi2


title: MLD_Day0_DES_Ut1


title: MLD_Day0_Oil_Ovi1


title: MLD_Day0_DES_Ovi2


title: MLD_Day0_DES_Vg1


title: MLD_Day0_DES_Ovi1


title: MLD_Day0_Oil_Ut3


title: MLD_Day0_DES_Vg3


status: Public on Jun 02 2009

city: Toronto

first: DCC

last: modENCODE

state: Ontario

address: MaRS Centre, South Tower, 101 College Street, Suite 800

country: Canada

institute: Ontario Institute for Cancer Research

hyb_protocol: For array hybridization, 2ug of the labeled DNA material was hybridized per chip for 18 h at 45C in a 3 M tetramethyl ammonium chloride/1x MES-based solution containing 100?g/mL Herring Sperm DNA, 0.02% Triton and 30pM of biotinylated control oligo B2 (Affymetrix). All reagents were from Invitrogen, except where noted otherwise.

scan_protocol: Scanned at 0.7 microns/pixel.

data_processing: Three biological replicates were hybridized for this experiment. The data from replicate arrays were quantile-normalized (Bolstad et al., 2003) and all arrays were scaled to a median array intensity given (e.g. 361). Processed data are obtained using following parameters: median value is 361   The sliding window approach (bandwidth 50) has been used to estimate RNA abundance (signal which is listed in column #2). It was found by calculating the median of all pairwise average PM-MM values, where PM is a perfect match and MM is a mismatch. Processed data are obtained using following parameters: bandwidth is 50   genome version is r5

label: Biotin

organism: Drosophila melanogaster

source_name: embryos,6-8 hr after egg laying (Celniker/RNA:232) extraction1_array1 channel_1

label_protocol: Double-stranded cDNA was treated with RNase H (Epicentre Technologies) and RNases A/T1 (Ambion), extracted by using a QIAquick PCR purification kit (Qiagen), and subjected to further fragmentation to 50-100 bp by DNase I (1 unit/ul; Epicentre Technologies; size distribution of fragmented DNA was verified on a 2% agarose gel).The fragmented cDNA was then end-labeled with 2.5mM biotinylated DNA labeling reagent (Affymetrix) by using 100 units of terminal deoxynucleotidyltransferase (TdT; Roche Diagnostics) in 1x TdT buffer (Roche Diagnostics) and 5 mM CoCl2 (Roche Diagnostics) for 2 h at 37C.

growth_protocol: ''This protocol was used for staging and collection of Drosophila melanogaster embryos by Dave Miller''''(Kaufman Lab, IU Bloomington) for transcriptome analysis by the Celniker group.''All Drosophila tissue collections were made from the sequenced ''D. melanogaster'' isogenic strain containing the mutations; ''yellow'' (y1), ''cinnabar'' (cn1), ''brown'' (bw1), and ''speck'' (sp1) [1].  Synchronized embryos were collected from population cages that were less than one week old and maintained at 24 C.  At least one ? two hour pre-lay was made prior to the start of timed collections each day. Embryos were collected for two hours on three hard egg lay collection plates made in 150 X 15 mm Petri dishes containing a substrate of 3.3% agar, 13% unsulfured molasses, and 0.15% Tegasept. The hard egg lay plates were completely covered with a thin layer of moist yeast paste (Fleischmann?s Baker?s Dry Yeast) and placed horizontally on a short 1cm raised Plexiglas bar in the bottom of each cage to avoid crushing flies.Staged embryos were passed through an 850 micron screen and collected on a 75 micron screen to remove adults and yeast paste.  Embryos were then dechorionated by treatment with 50% bleach (3% sodium hypochlorite), 0.2% sodium chloride, and 0.02% Triton-X-100 for five minutes.  Embryos were washed twice with 0.2% NaCl, 0.02% Triton buffer and split into two samples.  Most of the sample (approximately 95%) was rinsed with de-ionized water in a buchner funnel under mild vacuum, dried briefly, immediately frozen on dry ice and stored at -80? C for RNA preparations.  The small aliquot was transferred to a clean tube and fixed for staging.  Fixation was accomplished in 0.1 M Pipes (pH 6.9), 2 mM EGTA, 1 mM MgSO4, 4% paraformaldehyde, 0.1% glutaraldehyde (percentages are relative to aqueous volumes), and 50% heptane, as describe previously [2].  Samples were shaken for five minutes in the fixative, centrifuged briefly and the aqueous fraction was removed.  An equal volume of methanol containing 2 mM EGTA was added and the sample was shaken for five additional minutes.  Tissue was washed twice in methanol with 2 mM EGTA and saved at -80? C for the characterization of developmental stages.

extract_protocol:
RNA extraction: TRIzol procedure for Insects followed by DNase and RNeasyNote: This protocol is used to prepare RNA that will be used for microarray hybridization or RT-PCR.Materials	TRIzol® reagent: Invitrogen, cat. no. 15596-026 (100 ml) or 15596-018 (200 ml).	DNase/RNase-free water: Invitrogen cat. no. 10977.	75% ethanol, prepared with DNase/RNase-free water.	RNase-free DNase: Qiagen cat. no. 79254.	RNeasy® spin columns: RNeasy midi kit, Qiagen cat. no. 75144.	TenBroeck homogenizer: Bellco biotechnology cat no. 1982-10002.Procedure1.	Homogenize insect samples in TRIzol reagent using a TenBroeck tissue homogenizer. The sample volume should not exceed 10% of the volume of TRIzol reagent used for homogenization.2.	Incubate at room temperature for 5 minutes.  3.	Aliquot samples into 1.5 ml microcentifuge tubes.4.	Add 0.267 ml chloroform per ml of TRIzol used.5.	Shake tubes vigorously for 15 seconds.6.	Incubate at room temperature for 2 minutes.7.	Centrifuge for 15 minutes at 4degC at 12,000 x g.8.	Transfer the top (aqueous) phases to clean tubes.9.	Precipitate the RNA from the aqueous phase by adding 0.67 ml of isopropanol per ml of TRIzol used in step 5.10.	Invert tubes once to mix gently.11.	Incubate samples at room temperature for 10 minutes.12.	Centrifuge for 10 minutes at 4degC at 12,000 x g.13.	Remove the supernatant.14.	Wash RNA pellet once with 75% ethanol, using 0.7 ml per microcentrifuge tube.15.	Vortex briefly.16.	Centrifuge at 7,500 x g for 5 minutes at 4degC.17.	Let the pellet air dry for 10 minutes. Note: DO NOT let the pellet dry completely.18.	Dissolve in RNase-free water. 19.	Incubate at 37degC overnight to dissolve the RNA. Determine concentration using a Nanodrop® ND-1000 Spectrophotometer. RNA may be stored at -80degC at this point if desired.20.	Purify up to 1 mg of RNA on an RNeasy spin column, with DNase treatment, precisely as described in Qiagen's RNeasy Midi/Maxi Handbook, supplied with the RNeasy kit. Do the optional second wash on the column. Elute twice using DNase/RNase-free water.21.	Determine concentration by absorbance, using a Nanodrop® ND-1000 Spectrophotometer. If necessary, the RNA can be concentrated on a SpeedVac. Store at -80degC.
First-strand cDNA synthesis was performed by using SuperScript II reverse transcriptase in the reaction volume of 105 ul for 15 ug of starting RNA material. The RNA was mixed with random hexamers (83.3 ng/g mRNA), heated to 70C for 10 min, and cooled to 15C after which 5x SuperScript II First Strand buffer, DTT (10 mM), and dNTPs (0.5 mM) were added. SuperScript II was added after a 20-min incubation (200 units/ug RNA) followed by a 20-min ramp to 42C and 60-min incubation at 420C. SuperScript II was inactivated at 75C for 15 min.The second-strand cDNA was synthesized by addition of 50 units of Escherichia coli DNA ligase, 200 units of E. coli DNA polymerase I, 10 units of E. coli RNase H, and 0.2 mM dNTPs to the first-strand synthesis reaction at 16C for 2 h.

value: Embryo 6-8h

tag: genotype

tag: sex

value: Unknown