NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM967448 Query DataSets for GSM967448
Status Public on Jul 31, 2013
Title Salvia miltiorrhiza, replicates 1 & 2
Sample type genomic
 
Source name Salvia miltiorrhiza leaves
Organism Salvia miltiorrhiza
Characteristics tissue: leaves
family: Lamiaceae
order: Lamiales
Growth protocol Fresh or preserved leaf samples were obtained.
Extracted molecule genomic DNA
Extraction protocol Total DNA was extracted from leaves using a modification of the standard CTAB procedure.Approximately 0.5 g of leaves was ground with liquid nitrogen to a fine powder. The powder was dissolved in 5 ml of CTAB Buffer (3% CTAB, 100 mM Tris-HCL pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl), 1 ml of 10% PVP and 1.2 ml of 10% CTAB. Subsequently, the mixture was incubated at 60 °C for 1 h and centrifuged at 13,000 rpm for 10 min. The supernatant obtained was further purified with a double chloroform extraction followed by precipitation with 7.5 M ammonium acetate and 100% ethanol. The precipitated DNA was resuspended in sterile water and subsequent cleanup was performed by using the DNeasy® column of the DNeasy® Plant Mini Kit (Qiagen) following the protocol in the user manual.
Label Biotin
Label protocol The preparation of targets in all cases involved the double digestion of gDNA with AluI and HaeIII, and purification using the Qiaquick® PCR Purification Kit (Qiagen Inc.). Approximately 150 ng of purified digested DNA was labeled with Biotin-11-dUTP using the Biotin DecaLabelTM DNA Labeling Kit (Fermentas, ON, Canada). The reaction was incubated during 20 h and no further purification was performed.
 
Hybridization protocol The SDA slides were pre-hybridized for 45 min at 42°C in a pre-warmed solution containing 5X standard saline citrate (SSC), 0.1% sodium dodecyl sulphate (SDS), 1% bovine serum albumin (BSA) and 25% formamide. The slides were rinsed twice with sterile MilliQ water and immediately dried with an air gun.
The biotin-labelled targets (30 ng) were added to 17.5 μL of fresh 2X Hybridization buffer (250 μL of formamide, 250 μL of 10X SSC, 10 μL of 10% SDS), 0.5 µl of 1 mg/ml Human Cot1 DNA (Invitrogen); 0.5 µl of 5 mg/ml PolyA (Sigma-Aldrich) and 0.5 µl of 10 mg/ml salmon sperm DNA (Sigma-Aldrich). The mixture (made up to 35 µl with sterile water) was denatured at 100°C for 2 min and immediately applied onto the array under a 22 × 22-mm lifter slip (Grale Scientific, Victoria, Australia). The slides were then placed in waterproof, humidified hybridization chambers (Corning Incorporated Life Sciences) and incubated overnight in a 42°C water bath. After hybridization, the coverslips were removed and the slides were washed twice in 1 x SSC, 0.1% SDS at 40°C for 8 min, once in 0.1 x SSC, 0.1% SDS at 40°C for 8 min and once in 0.1 x SSC at room temperature for 5 min. Subsequently the slides were transferred to 500 mL of 6X SSPE-T buffer (0.9 M NaCl, 0.06 M NaH2PO4.H2O, 0.006 M EDTA, 0.005% Triton X-100, pH 7.4) without allowing them to dry.
The biotinylated DNA targets bound on the array were then labelled with fluorescent FluoroLink™ streptavidin-labelled Cy3 dye (Amersham Pharmacia, UK) using a biotin-streptavidin system. Briefly, 200 μL of a Detection solution (0.5 µL of 0.8 µg/µL streptavidin-labelled Cy3, 0.8 µl of 25 µg/µL BSA, made to 200 µL with 6X SSPE-T) was applied directly onto the array surface and a 25x60-mm lifter slip was placed over it to evenly distribute the solution on the array. The slides were placed in hybridization chambers, wrapped in aluminum foil and incubated at 37 °C for 40 min in the dark. Finally, the slides were washed thrice in 6X SSPE-T for 5 min and rinsed with deionized water before being dried with an air gun. All hybridizations were performed with four technical replicates (subarrays) and two biological replicates, for a total of eight data points per array feature.
Scan protocol Slides were scanned with a ScanArray Gx (PerkinElmer Life and Analytical Sciences, Downers Grove, IL) microarray scanner in conjunction with the supplied software. The slides were scanned with a resolution of 10 µm at 532 nm (Cy3, green laser) and at 50% photomultiplicator (PMT) gain whilst keeping background noise low. The scanned array was quantified using PerkinElmer ScanArray Express software v 2.0. The program individually quantified the signal intensity at each probe and normalized the data using the adaptive circle and LOWESS functions.
Description This Sample represents 2 biological replicates.
Data processing Probes which did not hybridize were automatically flagged by the scanning software and labelled as 'bad'. Manual flagging was used to remove spots displaying inconsistent hybridization such as 'donut' spots. 'Good' probes were accepted as having a mean 'signal-to-noise ratio' (SNR) value of greater than 7 in more than half of the technical replications. Data analysis included: Calulation of the mean signal-to-noise ratio for each feature between the four technical replicates. Normalization across the slides using the mean signal-to-noise ratio for all 285 spots in all hybridizations performed. Average of the biological replicates after normalization to produce a single value per feature.
 
Submission date Jul 16, 2012
Last update date Jul 31, 2013
Contact name Nitin Mantri
E-mail(s) nitin.mantri@rmit.edu.au
Organization name RMIT University
Department School of Science
Street address Plenty Road
City Bundoora
State/province Victoria
ZIP/Postal code 3083
Country Australia
 
Platform ID GPL15813
Series (1)
GSE39403 A gDNA microarray for genotyping Salvia species

Data table header descriptions
ID_REF
VALUE Normalized mean signal intensity

Data table
ID_REF VALUE
O17 6.184875833
A18 21.66620833
C18 21.70487
E18 20.6190925
G18 17.32482917
I18 14.12597583
K18 7.300711667
M18 16.82823583
O18 7.727633333
A19 18.21311
C19 12.13736333
E19 21.54520833
G19 5.061058333
I19 31.77799667
K19 14.672495
P17 4.35938
A16 27.42917167
C16 60.54957833
E16 22.60288167
G16 25.91397

Total number of rows: 285

Table truncated, full table size 4 Kbytes.




Supplementary file Size Download File type/resource
GSM967448_Salvia_miltiorrhiza_BR1_gain_50.txt.gz 12.0 Kb (ftp)(http) TXT
GSM967448_Salvia_miltiorrhiza_BR2_gain_50.txt.gz 40.7 Kb (ftp)(http) TXT
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap
External link. Please review our privacy policy.