|
| Status |
Public on Jan 16, 2008 |
| Title |
Peripheral blood_male_Pitta_rep2_Ex.set2&3 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral blood cells from pitta prakriti males
|
| Organism |
Homo sapiens |
| Characteristics |
total RNA from peripheral blood cells of second set of pitta prakriti males of age group 18-40yrs labeled with Cy3, and compared with values obtained in set 3.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Using EZ-RNA isolation kit (Biological Industries) as per manufacturers protocol.
|
| Label |
Cy3
|
| Label protocol |
Three samples of each prakriti were pooled. Experiments for male and female were carried out separately. Double stranded cDNA was synthesized from 15 µg (5 µg each sample) of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each pool of Vata, Pitta and Kapha purified cDNA was divided into two halves, one half labeled with Cy5 and other with Cy3 (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche) and labeled products were purified by Microarray Target Purification Kit (Roche).
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| |
|
| Hybridization protocol |
The two cRNA samples of each prakriti, one labeled with Cy3 and another with Cy5, were pooled together, precipitated, washed and air-dried. The dried pellet was dissolved in RNAase free water (Sigma). Hybridization solution was prepared by mixing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma) and added to the labeled product. This mixture was denatured at 65ºC and applied onto cDNA microarray slides. Inter prakriti cohybridization was carried out at 37ºC for 16 hrs.
|
| Scan protocol |
Microarray slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices), using both green and red lasers. The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices).
|
| Description |
Log2 normalized values of the analyzable spots commonly obtained between 2and 3 experimental sets, for the second biological replicate of Pitta males
|
| Data processing |
Analysis was carried out taking two experiments at a time, separately for male and female data. Background subtracted mean values were used for the analysis. All the negative values were removed and only those genes having positive values for all the samples were considered for further analysis. Values were log2 transformed and lowess normalization. Across array normalization was carried out using quantile normalization method. One-way analysis of variance was adopted to identify differentially expressed genes in all three categories. F-test was carried out in order to reject the hypothesis of equality of three group means. in order to investigate further the nature of differentially expressed genes; we have drawn strip charts and carried out pair wise comparisons using t-test. p-values obtained from ANOVA have been adjusted for multiplicity using Bonferroni method for controlling family wise error rate. Genes at p0.1 level of significance were considered to be significantly different in expression between all three groups.
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| Submission date |
May 23, 2007 |
| Last update date |
Jan 16, 2008 |
| Contact name |
Mitali Mukerji |
| E-mail(s) |
mitali@igib.res.in
|
| Phone |
91-11-29879302
|
| Organization name |
CSIR-Institute Of Genomics and Integrative Biology
|
| Department |
Functional Genomics and Molecular Medicine
|
| Lab |
319
|
| Street address |
Sukhdev Vihar, Mathura Road
|
| City |
New Delhi |
| State/province |
New Delhi |
| ZIP/Postal code |
110020 |
| Country |
India |
| |
|
| Platform ID |
GPL2829 |
| Series (1) |
| GSE7883 |
Expression profile of peripheral blood of healthy individuals phenotyped based on “Prakriti” concept of Ayurveda |
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