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Sample GSM319883 Query DataSets for GSM319883
Status Public on Dec 01, 2008
Title High AP3 producing Actinosynnema pretiosum (ATCC:31565) rifampicin mutant_Biolreplicate2_day2
Sample type mixed
 
Channel 1
Source name Rifampicin resistant mutant, derived from Actinosynnema pretiosum, cultured in YMG day 2
Organism Actinosynnema pretiosum
Characteristics Derived from Actinosynnema pretiosum (ATCC:31565) Resistant to 50ug/mL rifampicin
Growth protocol Spores from single colonies were cultured in Vegetative medium (5 g/l meat extract, 5/l g peptone, 5 g/l yeast extract, 2.5 g/l enzyme hydrolysate of casein, 20 g/l glucose, 1.5 g/l NaCl) for 2 days at 26oC. 2mL of seed culture was used to innoculate 320mL of YMG media (4 g/l yeast extract, 10 g/l malt extract, 4 g/l glucose, pH 7.2) Cultures were incubated at 26oC shekn at 190rpm. Samples were taken at day 2, 4, 6 and 8 of the culture. The samples were centrifuged at 3000g at 4oC for 15 mintues. The supernatent was used for Ansamitocin-P3 analysis and the volume of the cell pellet estimated(PCV) before freezing and storing at -80oC.
Extracted molecule total RNA
Extraction protocol DEPC treated water equvilent to 0.5 x volume of cell pellet was added to the cell pellet. After cell pellet has thawed, it is subjected to 5 more cycles of freezing thawing in liquid nitrogen and a 55oC waterbath respectively. The cell pellet is then treated with lysozyme (3.5 mg/ml) at 37oC for 20 min. The supernatent removed through centrifugation and the pellet mixed with Rnawiz (Ambion, USA) and RiboPureTM-Bacteria Kit (Ambion, USA) was used as per manufecturer's protocol. Optional Dnase cleanup step was also performed.
Label Cy3
Label protocol 10ug of total RNA was incubated with 2ug of randomer hexamer. Incubated at 70Co for 5 mintues and placed on ice. 1pmole of Cy3 labelled dUTP, low dT-dNTP mix Rnase inhibitor and 2uL of RevertAidTM H- Minus M- MuLV Reverse Transcriptase (Fermentas Life Sciences, MD) was added. Mixture is incubated at room temperature for 10 minutes folloed by 42Co for 2 hours
10 µg of total RNA were primed with 2 µl of 100 µM random DNA primer (N9) at 70°C for 5min, then reversed transcribed at 42°C for 1 h in the presence of 400 U RevertAidTM H- Minus M- MuLV Reverse Transcriptase (Fermentas Life Sciences, MD), and 25 µM each dATP, dCTP, dGTP, with 12.5 µM dTTP, and 25 µM Cy3-labelled dUTP.
 
Channel 2
Source name Actinosynnema pretiosum sheared genomic DNA
Organism Actinosynnema pretiosum
Characteristics Strain: ATCC number:31565
Growth protocol Spores from single colonies were cultured in Vegetative medium (5 g/l meat extract, 5/l g peptone, 5 g/l yeast extract, 2.5 g/l enzyme hydrolysate of casein, 20 g/l glucose, 1.5 g/l NaCl) for 2 days at 26oC. 2mL of seed culture was used to innoculate 320mL of YMG media (4 g/l yeast extract, 10 g/l malt extract, 4 g/l glucose, pH 7.2) Cultures were incubated at 26oC shekn at 190rpm. Samples were taken at day 2, 4, 6 and 8 of the culture. The samples were centrifuged at 3000g at 4oC for 15 mintues. The supernatent was used for Ansamitocin-P3 analysis and the volume of the cell pellet estimated(PCV) before freezing and storing at -80oC.
Extracted molecule genomic DNA
Extraction protocol DEPC treated water equvilent to 0.5 x volume of cell pellet was added to the cell pellet. After cell pellet has thawed, it is subjected to 5 more cycles of freezing thawing in liquid nitrogen and a 55oC waterbath respectively. The cell pellet is then treated with lysozyme (3.5 mg/ml) at 37oC for 20 min. The supernatent removed through centrifugation and the pellet mixed with Rnawiz (Ambion, USA) and RiboPureTM-Bacteria Kit (Ambion, USA) was used as per manufecturer's protocol. Optional Dnase cleanup step was also performed.
Label Cy5
Label protocol Actinosynnema pretiosum genomic DNA was sheared by sonicating on ice with the Vibra-CellTM fitted with a 3mm probe (Sonics and Materials, CT) for 1 min. 0.1ug of sheared gDNA was incubated with 300µg/mL random DNA octomers at 96Co for 5 mintues then placed on ice. It was then incubated with 50U of klenow fragments (New England Biolabs, USA), 120µM each of dATP, dCTP,dGTP, 60µM of dTTP and 40µM of Cy5-labelled dUTP at 37oC for 2 hours.
 
 
Hybridization protocol Slides were pre-hybridized with 5x saline sodium citrate buffer(SSC), 50% deionised formamide, 0.1% sodium dodecyl sulphate(SDS) and 1 % Bovine serum albumin for 2 hour at 42oC. Labelled cDNA and gDNA was cleaned up using mineluate columns per manufacture’s protocol (Qiagen, USA). Hybridisation mixture comprising of labelled cDNA from 10ug of total RNA, 0.1ug of labelled gDNA, 5 x SSC, 25% deionised formamide and 0.1% SDS were heated at 95oC for 5 minutes, cooled on ice before sample was applied to the microarray slides and enclosed in hybridisation chambers for 16 hours at 42oC. Cover slips of the microarray were firsted removed in SSC and 0.2% SDS before being washed in twice in 2 x SSC for 5 mintues each wash, two five minutes wash in 0.1 x SSC with 0.1%SDS and final two washes of 5 mintues each in 0.1x SSC. Slides were subsequently dried by centrifuging at 129g for 3 minutes.
Scan protocol Slides were scanned on a Axon GenePixÔ 4000B scanner and images were analysed using GenePix Pro 4.1. Expression data was exported into microsoft Excel for normalisation and further analysis
Description Biological replicate : r50D1 rifampicin resistant clone derived from Actinosynnema pretiocum (ATCC:31565) day 2 sample vs gDNA reference 2 technical repeats
Data processing Zero or negative value background corrected readings were assigned a value of one. Quantile normalization was carried out based on the method described in Mehra et al. {Mehra, 2006 157 /id}. Briefly, log2(cDNA/gDNA) was first calculated for each data point, before the log ratio of each array was sorted in ascending order and ranked accordingly. The “reference” array was generated by taking the median value of each rank. To normalize the data, percentile values from each of the array to be normalized was replaced by the corresponding value from the reference array before unsorting back to the original order. Technical replicates were averaged and standard deviation was calculated for log intensity ratios for each probe. Probe with log intensity ratios more then 1 standard deviation from the average were treated as outliers and discarded. Technical replicates were then averaged again. Each chip was then scale normalized {Quackenbush, 2002 301 /id} by subtracting the 16s log ratio from each data point.
 
Submission date Sep 11, 2008
Last update date Sep 11, 2008
Contact name Peter Nissom
E-mail(s) pnissom@gmail.com
Organization name Bioprocessing Technology Institute
Street address 20 Biopolis Way
City Singapore
ZIP/Postal code 138668
Country Singapore
 
Platform ID GPL7284
Series (1)
GSE12739 Transcriptional profile asm gene cluster of high Ansamitocin P3 producing mutants derived from A. pretiosum

Data table header descriptions
ID_REF
VALUE normalized log2 ratio (Cy3/Cy5) representing test/reference

Data table
ID_REF VALUE
16S rRNA_1 0
16S rRNA_2 -1.571279071
16S rRNA_3 1.173985944
16S rRNA_4 0.739554783
aroF1 -0.891000973
aroF2 1.20637974
asm1 -0.373965064
asm10 0.650945694
asm12 -0.426599288
asm13 -0.799078804
asm14 -1.908335002
asm16 -1.248218214
asm17 -0.838681105
asm19 -0.157859795
asm2 -2.862964607
asm20 -0.125504698
asm21_1 -1.247808369
asm21_2 -1.282481947
asm21_3 -4.575459818
asm22 -0.963425292

Total number of rows: 69

Table truncated, full table size 1 Kbytes.




Supplementary file Size Download File type/resource
GSM319883_r50D1_E_y2_2008-02-19_0009b.txt.gz 64.5 Kb (ftp)(http) TXT
GSM319883_r50D1_E_y2_2008-02-19_0010b.txt.gz 64.6 Kb (ftp)(http) TXT
Processed data included within Sample table

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