|
| Status |
Public on Oct 05, 2016 |
| Title |
Spleen of Leishmania-infected Balb/c mice [Ba-rL7] |
| Sample type |
RNA |
| |
|
| Source name |
Spleen of Leishmania-infected Balb/c mice
|
| Organism |
Mus musculus |
| Characteristics |
strain: Balb/c
age: 14-15 weeks old
infected with: 10^6 stationary-phase L. infantum promastigotes via tail vein
tissue: spleen
|
| Treatment protocol |
24 out of the 47 mice were infected with 10^6 stationary-phase L. infantum promastigotes via tail vein.
|
| Growth protocol |
47 BALB/c (purchased to Charles River Laboratories, France) mice (14-15 weeks old) were used in this study. Mice were randomly separated in two groups: (i) 23 control mice and (ii) 24 mice that were infected with 10^6 stationary-phase L. infantum promastigotes via tail vein. Mice were euthanized by cervical dislocation and spleens were removed and immediately stored in RNAlater at -70ºC (Sigma-Aldrich, St. Louis, USA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation from spleens (9-11 mg) was performed by cell disruption using FastPrep® System (ProScientific, Cedex, France) and Lysing Matrix D (MP Biomedicals, Solon, USA) in TRI-Reagent (Sigma-Aldrich, St. Louis, USA). RNeasy Mini Kit (Qiagen) was subsequently used for mRNA enrichment following manufacturer’s instructions
|
| Label |
FAM
|
| Label protocol |
The reverse transcription was performed with 2 ug of RNA with random hexamers using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA) according to manufacturer’s instructions.
Real-time PCR and fluorescence detection were performed using QuantStudio 12K Flex Real-Time PCR System (Life Technologies, Carlsbad, CA) following manufacturer’s instructions, using Custom TaqMan OpenArray Real-Time PCR Plates
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| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
TEST
|
| Data processing |
Cq values from the 47 samples analyzed were exported for normalization using three different algorithms: geNorm (qBasePLUS software (Biogazelle, Gent, Belgium)), NormFinder (http://moma.dk/normfinder-software) and RefFinder (http://fulxie.0fees.us/?type=reference).
The sample data table includes the normalized data using Il2rg+Itgb2 as reference genes, as identified and validated in our paper.
The 'Polr2a_Tbp_normalized.tx't includes the data normalized using Polr2a+Tbp as reference genes, two reference genes traditionally used in the literature for this model.
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| |
|
| Submission date |
Apr 27, 2016 |
| Last update date |
Oct 05, 2016 |
| Contact name |
Emma Carmelo |
| E-mail(s) |
ecarmelo@ull.edu.es
|
| Phone |
639648290
|
| Organization name |
University of La Laguna
|
| Department |
University Institute of Tropical Diseases and Public Health
|
| Street address |
C/ Astrofisico Francisco Sanchez s/n, Campus de Anchieta
|
| City |
La Laguna |
| State/province |
SANTA CRUZ DE TENERIFE |
| ZIP/Postal code |
38296 |
| Country |
Spain |
| |
|
| Platform ID |
GPL21778 |
| Series (1) |
| GSE80709 |
The Challenge of Stability in High-Throughput Gene Expression Analysis: Comprehensive Selection and Evaluation of Reference Genes for BALB/c Mice Spleen Samples in the Leishmania infantum Infection Model. |
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