|
| Status |
Public on Jul 01, 2016 |
| Title |
Sarupat (Resistant) race uninfected control- replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Midgut tissue of fifth instar silkworm larvae
|
| Organism |
Bombyx mori |
| Characteristics |
race: Sarupat
bmnpv resistance status: resistant
bmnpv infection status: uninfected
tissue: Midgut
developmental stage: Silkworm larvae after fourth moult out
|
| Treatment protocol |
Virus inoculations: B. mori multiple nucleopolyhedrovirus stock was maintained at this laboratory and used as viral inoculum. The viral inoculum was prepared by counting the number of viral polyhedral in a Neubauer chamber. The oral inoculation of BmNPV occlusion bodies was carried out in healthy newly moulted ‘0 day’ fifth instar larvae (first day after 4thmoult) of Sarupat and CSR-2 races with viral dosage of 40,000 polyhedral inclusion bodies (PIB) per larva. Three replications containing twenty-five silkworms were maintained for each silkworm race. Similarly, the uninoculated control batches were reared separately under disease free environment. Silkworms feeding on BmNPV-free mulberry leaves were placed in labelled boxes until feeding was complete and then transferred to a controlled room where they remained until the end of the experiment.
|
| Growth protocol |
Selection of silkworm races The silkworm B. mori races viz., Sarupat and CSR-2 were selected for the study, as these are known to be most resistant and most susceptible to BmNPV. These two silkworm races were used for the microarray as well as for quantification and gene expression analysis using qPCR.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was done by using QIAGEN RNeasy mini kit (Cat#74106) as per the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
The samples were labeled using Agilent Quick-Amp labeling kit (Part number: 5190-0424). 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.
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| |
|
| Hybridization protocol |
1650ng of labeled cRNA were hybridized on the array (AMADID: 66047) using the Agilent Gene Expression Hybridization kit (Part Number 5190-0404) 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) at 5 micron resolution.
|
| Description |
Gene expression in uninfected silkworms
|
| Data processing |
Data extraction from Images was done using Feature Extraction software and Normalization of the data was done in GeneSpring GX: 75th percentile shift method
|
| |
|
| Submission date |
Jul 22, 2015 |
| Last update date |
Jul 01, 2016 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL20728 |
| Series (1) |
| GSE71240 |
Genome wide microarray based expression profiles associated with BmNPV resistantance and susceptibility in Indian silkworm races of Bombyx mori |
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