|
| Status |
Public on Sep 15, 2015 |
| Title |
Lame132 Area3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Acid_follic_B12
|
| Organism |
Bos taurus |
| Characteristics |
compound added: Folic Acid (320mg) and vitamin B12 (10mg)
|
| Treatment protocol |
Cows were given folic acid and vitamin B12 (treatment group) or physiological saline (control group) three weeks before calving till 57±2 days post-calving.
|
| Growth protocol |
Approximately 66 ± 2 hours after the second injection of PGF2 alpha, granulosa cells populations were collected by transvaginal ovarian pick-up (OPU) of the preovulatory follicle. Only follicles with a diameter equal to or greater than 12 mm were collected. In the absence of preovulatory follicle on day 57 ± 3, a second OPU was performed on day 71 ± 3. OPU was performed under the effect of an epidural injection of lidocaine, with a 19 gauge needle 1 - ½ inch and a vacuum pump. Granulosa cells were sorted under a stereomicroscope. To isolate the cells from the follicular fluid, a centrifugation at 800 × g was performed for 2 minutes at room temperature before storage. The follicular fluid was then decanted, measured and quickly immersed in liquid nitrogen. The pellet was suspended in 500 μl of PBS at 4 °C. After centrifugation for 2 minutes at 800 × g, the supernatant was removed and granulosa cells frozen in liquid nitrogen. Samples were stored at -80 °C until used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
This document applies to the total RNA extraction using the PicoPure RNA Isolation Kit from Molecular Devices. The PicoPure RNA Isolation Kit enables to recover total cellular RNA from pico-scale samples. Researchers can obtain high recoveries of total cellular RNA from as little as a single cell to samples with up to 100 ug of RNA. RNA extraction from larger samples should be done using another kit or technique.
|
| Label |
Cy5
|
| Label protocol |
This procedure describes the amplification of RNA using the RiboAmpb HSPlus RNA Amplification kit (Molecular devices). This procedure may be required for microarray experiments with extremely limited amounts of starting material. The RiboAmpb HSPlus RNA Amplification Kit enables the production of microgram quantities of antisense RNA (aRNA) from picogram quantities of total cellular RNA. The RiboAmpb HSPlus Kit achieves high yields of aRNA with a proprietary linear amplification process using double-stranded cDNA as template in a two round T7 RNA polymerase catalyzed amplification.
This document describes the procedure used for labelling aRNA using the Universal Linkage System (ULS) by Kreatech for Gene Expression studies using microarray slides from Agilent. ULS is a non-enzymatic protocol that allows direct labeling of unmodified, amplified RNA. ULS will react with unmodified, amplified RNA and label it by formation of a coordinative bond on the N7 position of guanine. Fluorophore Cy3 and Cy5 are linked via a spacer to the ULS molecule.
|
| |
|
| Channel 2 |
| Source name |
CTR
|
| Organism |
Bos taurus |
| Characteristics |
compound added: Physiological saline 0.9%
|
| Treatment protocol |
Cows were given folic acid and vitamin B12 (treatment group) or physiological saline (control group) three weeks before calving till 57±2 days post-calving.
|
| Growth protocol |
Approximately 66 ± 2 hours after the second injection of PGF2 alpha, granulosa cells populations were collected by transvaginal ovarian pick-up (OPU) of the preovulatory follicle. Only follicles with a diameter equal to or greater than 12 mm were collected. In the absence of preovulatory follicle on day 57 ± 3, a second OPU was performed on day 71 ± 3. OPU was performed under the effect of an epidural injection of lidocaine, with a 19 gauge needle 1 - ½ inch and a vacuum pump. Granulosa cells were sorted under a stereomicroscope. To isolate the cells from the follicular fluid, a centrifugation at 800 × g was performed for 2 minutes at room temperature before storage. The follicular fluid was then decanted, measured and quickly immersed in liquid nitrogen. The pellet was suspended in 500 μl of PBS at 4 °C. After centrifugation for 2 minutes at 800 × g, the supernatant was removed and granulosa cells frozen in liquid nitrogen. Samples were stored at -80 °C until used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
This document applies to the total RNA extraction using the PicoPure RNA Isolation Kit from Molecular Devices. The PicoPure RNA Isolation Kit enables to recover total cellular RNA from pico-scale samples. Researchers can obtain high recoveries of total cellular RNA from as little as a single cell to samples with up to 100 ug of RNA. RNA extraction from larger samples should be done using another kit or technique.
|
| Label |
Cy3
|
| Label protocol |
This procedure describes the amplification of RNA using the RiboAmpb HSPlus RNA Amplification kit (Molecular devices). This procedure may be required for microarray experiments with extremely limited amounts of starting material. The RiboAmpb HSPlus RNA Amplification Kit enables the production of microgram quantities of antisense RNA (aRNA) from picogram quantities of total cellular RNA. The RiboAmpb HSPlus Kit achieves high yields of aRNA with a proprietary linear amplification process using double-stranded cDNA as template in a two round T7 RNA polymerase catalyzed amplification.
This document describes the procedure used for labelling aRNA using the Universal Linkage System (ULS) by Kreatech for Gene Expression studies using microarray slides from Agilent. ULS is a non-enzymatic protocol that allows direct labeling of unmodified, amplified RNA. ULS will react with unmodified, amplified RNA and label it by formation of a coordinative bond on the N7 position of guanine. Fluorophore Cy3 and Cy5 are linked via a spacer to the ULS molecule.
|
| |
|
| |
| Hybridization protocol |
This document applies to the hybridization of microarray slides 4x44K printed with oligonucleotides by Agilent for all EmbryoGENE related projects.
|
| Scan protocol |
According to Tecan scanning protocol.
|
| Description |
Array 1
|
| Data processing |
LOESS normalized within array, background subtracted and quantile normalized between array data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used.
|
| |
|
| Submission date |
Aug 13, 2015 |
| Last update date |
Sep 15, 2015 |
| Contact name |
Annie Gagnon |
| E-mail(s) |
annie.gagnon.25@ulaval.ca
|
| Organization name |
Laval University
|
| Street address |
2440 boul. Hochelaga
|
| City |
Quebec |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 0A6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13226 |
| Series (1) |
| GSE72038 |
Effects of Intramuscular Administration of Folic Acid and Vitamin B12 on Granulosa Cells Gene Expression in Postpartum Dairy Cow |
|
