|
Status |
Public on Oct 25, 2016 |
Title |
TH_T |
Sample type |
RNA |
|
|
Source name |
macrophages cells
|
Organism |
Bos indicus |
Characteristics |
tissue: Blood time point: 6 hr infection infection: Mycobacterium bovis
|
Treatment protocol |
Monocyte cells were harvested for treatments of M.Bovis 6 h post stimulation and stored at -80 0C in 1 ml of Trizol1 reagent (Invitrogen Ltd.).
|
Growth protocol |
Blood from the jugular vein was collected into lithium heparin and in 15 ml clean centrifuge tube, equal amount of blood and Histopaque 1077 (Sigma-Aldrich Ireland Ltd., Dublin, Ireland) centri. at 850 × g for 20 min., buffy coats were harvested, diluted 1:3 in PBS, centri. at 1200 × g for 30 min. PBMC were then washed twice in PBS at 500 × g for 10 min and in RPMI 1640 with 10% foetal calf serum and penicillin/streptomycin) placed at 5 × 106 PBMC in 50 ml teflon flasks. Non-adherent cells were removed by washes with pre-warmed PBS, and adhered monocyte cells were cultured for 6 days at 370C, with 5% CO2 to obtain MDMs.
|
Extracted molecule |
total RNA |
Extraction protocol |
RNA isolation was performed using Qiagen RNeasy Mini Kit (CAT# 74106 ) following the manufacturer's recommendations.
|
Label |
Cy3
|
Label protocol |
The samples were labeled using Agilent Quick-Amp labeling kit (Part number: 5190-0442). 500ng of total RNA was reverse transcribed using oligo dT based method. mRNA was primed with oligo dT primer tagged to T7 promoter sequence and converted into double stranded cDNA using MMLV-RT reverse transcriptase. Further, in the same reaction cDNA was in-vitro transcribed to cRNA using T7 RNA polymerase enzyme. During cRNA synthesis, Cy3 labeled Cytosine nucleotide was incorporated into the newly synthesized strands. Labeled cRNA thus obtained was cleaned up using Qiagen RNeasy columns (Qiagen, Cat No: 74106). Concentration and amount of dye incorporated were determined using Nanodrop ND-1000.
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Hybridization protocol |
Samples that pass the QC for specific activity were taken for hybridization. 600ng of labeled cRNA were hybridized on the array (AMADID: 29411) using the Gene Expression Hybridization kit (Part Number 5190-0404; Agilent) in Sure hybridization Chambers (Agilent) at 65ºC for 16 hours. Hybridized slides were washed using wash buffers (Part No: 5188-5327; Agilent)
|
Scan protocol |
scanned using the Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D)
|
Description |
Gene Expression after 6 hr infection of M.Bovis in cattle blood
|
Data processing |
Images were quantified using Feature Extraction Software ( Agilent). Feature extracted raw data was analyzed using GeneSpring GX Version software from Agilent. Normalization of the data was done in GeneSpring GX using the 75th percentile shift
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|
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Submission date |
Oct 15, 2015 |
Last update date |
Oct 25, 2016 |
Contact name |
SANJEEV KUMAR SHUKLA |
E-mail(s) |
sanjeevcloning@gmail.com
|
Phone |
+919457273980
|
Organization name |
Indian Veterinary Research Institute
|
Department |
Division of Animal Genetics
|
Lab |
Blood Biochemical Polymorphism Laboratory
|
Street address |
IZATNAGAR
|
City |
Bareillly |
State/province |
UTTAR PRADESH |
ZIP/Postal code |
243122 |
Country |
India |
|
|
Platform ID |
GPL21036 |
Series (1) |
GSE74050 |
Developing gene signature against Mycobacterium bovis infection in Cattle |
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