|
| Status |
Public on Oct 04, 2006 |
| Title |
BCC-P36-PB3 - mAdbID:59807 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PBMC Pooled Donor Reference Sample
|
| Organism |
Homo sapiens |
| Characteristics |
Cell type: peripheral blood mononuclear cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sample Extraction Protocol
Other: RNA isolation from skin samples and modified RNA amplification for microarrays and Taqman real time PCR Total RNA was isolated using RNeasy minikits (Qiagen, Germantown, MD). Amplified antisense RNA (aRNA) was prepared from total RNA (0.5-3 ug) according to a modified version (see modification to original protocol, A-C below) of the protocol previously described by us (Wang et al Nature biotech 2000;17(4):457-459). The modifications introduced for total RNA isolation were the following: A) Tissue was homogenized through a 15ml disposable small tissue grinder (Fisher Scientific cat# 06-434A) containing originally 150ul of lysis buffer (RLT buffer + 2ul- betaME) and subsequently an additional 200 ul of lysis buffer to complete tissue homogenization and lysis. The lysate was then transferred to a QIAshreddder spin column and centrifuged at for 2 minutes at max speed. B) To remove proteins that interfered with RNA isolation, 590ul double-distilled water were added to the homogenate followed by 10ul PROTEINASE K solution (Qiagen) and mixed thoroughly by pipetting. The homogenate was then incubated at 55C for 10 minutes and centrifuged for 3 min at 13000 rpm at room temp. The supernatant (approximately 900ul) was then combined and mixed well with 0.5 volumes (usually 450 ul) of ethanol (96-100%) into a new Rnase-Dnase free tube mixed well by pipetting. To Separate RNA from DNA. 700ul of the sample (including any precipitate that may have formed) were transferred into a RNeasy mini column placed in a 2ml collection tube. Total RNA was isolated according to the manufacturer protocol. The modifications introduced for Antisense RNA amplification were the following: C) First strand cDNA synthesis was accomplished in the presence of 1ul SUPERase÷In (Ambion Cat# 2696 stored at Ÿ20 øC) instead of Rnasein Ambion Cat# 2696 stored at Ÿ20 øC) and ThermoScript RT (Gibco BRL Cat# 12236-022) instead of Superscript . One microliter of BSA (2ug/ul) was added as a carrier to each samples to increase RNA yield. D) A higher concentration of 1st round of amplification material was achieved by resuspending antisense RNA into « (11 ul) of original procedure volume and taking all of 1st round amplification-material to the second round of amplification. Quality of RNA was tested by Agilent technologies and spectophotometry (OD). Antisense RNA was either used for probe preparation and microarray analysis or for Taqman real time PCR. For microarray analysis test samples were labeled with Cy5-dUTP (Amersham, Piscataway, NJ) while the reference sample (pooled normal donor PBMC) was labeled with Cy3-dUTP. Test-reference sample pairs were mixed and co-hybridized to 17K-cDNA microarrays generated in our lab (gal file http://nciarray.nci.nih.gov/gal_files/gal_custom_current.shtml, Hs-CCDTM17.5k-1px.gal
|
| Label |
cy3
|
| Label protocol |
Cy3 Sample Labeling Protocol
Other: Target labeling by reverse transcription: 4ul First strand buffer 1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in 250ul DEPC H2O) 2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP) 2ul Cy-dUTP (1mM Cy3) 2ul 0.1 M DTT 1ul RNasin 3-6ug amplified mRNA in 7 ul DEPC H2O Mix well and heat to 65C for 5min then cool down to 42C. Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize the pH. Add 35ul of 1xTE. Target clean up: Prepare Bio-6 column and run target solution through it. Collect flow through and add 200ul 1xTE. Concentrate target to ~20ul using a microcon YM-30 column (Millipore Cat# 42410).
|
| |
|
| Channel 2 |
| Source name |
BCC-P36-PB3
|
| Organism |
Homo sapiens |
| Characteristics |
Tissue: skin
Disease state: basal cell carcinoma
|
| Treatment protocol |
Treatment type: placebo
Agent: vehicle cream
Treatment time: 4 days
Other: Cohort was treated twice a day for 4 days for a total of 4 doses.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sample Extraction Protocol
Other: RNA isolation from skin samples and modified RNA amplification for microarrays and Taqman real time PCR Total RNA was isolated using RNeasy minikits (Qiagen, Germantown, MD). Amplified antisense RNA (aRNA) was prepared from total RNA (0.5-3 ug) according to a modified version (see modification to original protocol, A-C below) of the protocol previously described by us (Wang et al Nature biotech 2000;17(4):457-459). The modifications introduced for total RNA isolation were the following: A) Tissue was homogenized through a 15ml disposable small tissue grinder (Fisher Scientific cat# 06-434A) containing originally 150ul of lysis buffer (RLT buffer + 2ul- betaME) and subsequently an additional 200 ul of lysis buffer to complete tissue homogenization and lysis. The lysate was then transferred to a QIAshreddder spin column and centrifuged at for 2 minutes at max speed. B) To remove proteins that interfered with RNA isolation, 590ul double-distilled water were added to the homogenate followed by 10ul PROTEINASE K solution (Qiagen) and mixed thoroughly by pipetting. The homogenate was then incubated at 55C for 10 minutes and centrifuged for 3 min at 13000 rpm at room temp. The supernatant (approximately 900ul) was then combined and mixed well with 0.5 volumes (usually 450 ul) of ethanol (96-100%) into a new Rnase-Dnase free tube mixed well by pipetting. To Separate RNA from DNA. 700ul of the sample (including any precipitate that may have formed) were transferred into a RNeasy mini column placed in a 2ml collection tube. Total RNA was isolated according to the manufacturer protocol. The modifications introduced for Antisense RNA amplification were the following: C) First strand cDNA synthesis was accomplished in the presence of 1ul SUPERase÷In (Ambion Cat# 2696 stored at Ÿ20 øC) instead of Rnasein Ambion Cat# 2696 stored at Ÿ20 øC) and ThermoScript RT (Gibco BRL Cat# 12236-022) instead of Superscript . One microliter of BSA (2ug/ul) was added as a carrier to each samples to increase RNA yield. D) A higher concentration of 1st round of amplification material was achieved by resuspending antisense RNA into « (11 ul) of original procedure volume and taking all of 1st round amplification-material to the second round of amplification. Quality of RNA was tested by Agilent technologies and spectophotometry (OD). Antisense RNA was either used for probe preparation and microarray analysis or for Taqman real time PCR. For microarray analysis test samples were labeled with Cy5-dUTP (Amersham, Piscataway, NJ) while the reference sample (pooled normal donor PBMC) was labeled with Cy3-dUTP. Test-reference sample pairs were mixed and co-hybridized to 17K-cDNA microarrays generated in our lab (gal file http://nciarray.nci.nih.gov/gal_files/gal_custom_current.shtml, Hs-CCDTM17.5k-1px.gal
|
| Label |
cy5
|
| Label protocol |
Cy5 Sample Labeling Protocol
Other: Target labeling by reverse transcription: 4ul First strand buffer 1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in 250ul DEPC H2O) 2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP) 2ul Cy-dUTP (1mM Cy5) 2ul 0.1 M DTT 1ul RNasin 3-6ug amplified mRNA in 7 ul DEPC H2O Mix well and heat to 65C for 5min then cool down to 42C. Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize the pH. Add 35ul of 1xTE. Target clean up: Prepare Bio-6 column and run target solution through it. Collect flow through and add 200ul 1xTE. Concentrate target to ~20ul using a microcon YM-30 column (Millipore Cat# 42410).
|
| |
|
| |
| Hybridization protocol |
Sample Hybrization Protocol
Wash procedure: Washing: 1. Wash with 2x SSC + 0.1% SDS to remove coverslip. 2. Wash with 1x SSC for 1 min. 3. Wash with 0.2x SSC for 1 min. 4. Wash with 0.05x SSC for 10-20 seconds. 5. Centrifuge slide at 80-100 g for 3 min.
Other: Hybridization: Combine Cy3 and Cy5 labeled target (adjust the color to purple) and complete dry the sample by speed vacuum . Add 37ul of the following mixture to the dried target (for 20x40mm slide): 1ul 50x Denhardtüs blocking solution (Sigma Cat# 2532) 1ul poly dA (8mg/ml Pharmacia Cat# 27-7988-01) 1ul yeast tRNA (4mg/ml Sigma Cat# R8759) 10ul Human Cot I DNA (1mg/ml Gibco BRL Cat# 15279-011) 3ul 20X SSC 1ul 10% SDS 20ul of DEPC H2O Heat for 2 min at 99oC and Cool to RT. Apply target mixture to array slide, add coverslip, place in humidified hyb chamber, and hybridize at 65oC over night.
|
| Description |
mAdb experiment ID: 59807
|
| Data processing |
After background correction and removal of flagged values, log base 2 expression ratios were median centered and linear transformed to obtain the log and linear values given in the data table.
|
| |
|
| Submission date |
Jun 21, 2006 |
| Last update date |
Oct 04, 2006 |
| Contact name |
Francesco Maria Marincola |
| E-mail(s) |
fmarincola@sidra.org
|
| Phone |
301-793-8210
|
| Organization name |
Sidra Medical and Research Center
|
| Street address |
Al Nasr Tower, AL Corniche Street, PO Box 26999
|
| City |
Doha |
| ZIP/Postal code |
PO Box 26999 |
| Country |
Qatar |
| |
|
| Platform ID |
GPL3895 |
| Series (1) |
| GSE5121 |
Imiquimod treated Basal Cell Carcinoma |
|
| Data table header descriptions |
| ID_REF |
NCI mAdb well id plus replicate number |
| VALUE |
same as UNF_VALUE but with flagged values removed |
| RATIO |
Calibrated Ratio (CY5 channel/CY3 channel) |
| Slide_block |
Array block location |
| Slide_column |
Array column location |
| Slide_row |
Array row location |
| CY5_mean |
Red Channel Sample mean Signal (Background Subtracted) |
| CY5_SD |
Red Channel Sample Standard Deviation |
| CY5_BKD_median |
Red Channel Sample median Background Level |
| CY5_BKD_SD |
Red Channel Sample Background Standard Deviation |
| CY3_mean |
Green Channel Sample mean Signal (Background Subtracted) |
| CY3_SD |
Green Channel Sample Standard Deviation |
| CY3_BKD_median |
Green Channel Sample median Background Level |
| CY3_BKD_SD |
Green Channel Sample Background Standard Deviation |
| Flag |
Quality flag 0->good, -50->Not found, -100->Bad |
| UNF_VALUE |
log2 (Cy5/Cy3) |
