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Status |
Public on Feb 03, 2014 |
Title |
Transiently transfected hEEC from biopsy I with MIMIC_30d miRNA |
Sample type |
RNA |
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Source name |
primary hEEC_miR-30d mimic
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Organism |
Homo sapiens |
Characteristics |
cell type: primary hEEC derived from healthy human endometrial biopsies at LH+0 transfected with: miR-30d mimic for 72hr
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Growth protocol |
When hEEC cultures reached 50% confluency cells were transiently transfected with 50nM of either miR-30d mimic or Scramble miRNA using HiPerfect as recommended by the manufacturer (Qiagen, Valencia, CA, USA). After 72 hours post-transfection, cells were subjected to RNA extraction.
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Extracted molecule |
total RNA |
Extraction protocol |
To ensure that miRNA fraction will be recovered we performed RNA extraction using the miRNeasy Kit (Qiagen, Valencia, CA, USA). RNA extracted was quantified using a NanoDrop (Thermo Fisher Scientific Inc, MA, USA) spectrophotometer and the quality of RNA samples was assesed using a Nano LabChip BioAnalyzer 2100 (Agilent Technologies Inc, DE, USA).
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Label |
Cy3-CTP
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Label protocol |
100ng of total RNA were mixed with spike and mixed with T7 promoter primer for 10' at 65C then cooled at 4C. Next to cDNA synthesis, First strand buffer, DTT, dNTP and RNAse block mix were added and incubated for 2 hours at 40C followed by 15' at 70C. Next for cRNA synthesis, transcription buffer, DTT, NTP, T7 RNA polymerase and Cy3-CTP were added and incubated for 2 hours at 40C and cooled for 5'. Then cRNA was purified according to Qiagen RNEasy kit protocol specifications. Yield and [cRNA] stimated. Then 1.65 ug de cRNA was used to be fragmented in presence of blocking agent and fragmentation buffer for 30' at 60C and cooled. Then sample was mixed with hybridization buffer.
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Hybridization protocol |
1.65 ug of cRNA was used to be fragmented in presence of blocking agent and fragmentation buffer for 30' at 60C and cooled. Then sample was mixed with hybridization buffer.
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Scan protocol |
Microarrays slides were washed and scanned in Genepix Personal 4100A Scanner and scanned in Genepix Pro Software with a spot resolution of 5um in the green channel.
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Description |
MIMIC_30d_I each sample was previous tested for total RNA quality using total Nano RNA Labchip in an agilent bioanalyzer and with RIN above 9.0
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Data processing |
Median intensity value of each spot data was log2 transformed and normalized using R software and libraries from bioconductor database. Next, replicated probes were merged by mean using GEPAS.
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Submission date |
May 08, 2013 |
Last update date |
Feb 03, 2014 |
Contact name |
Juan M Moreno-Moya |
E-mail(s) |
jmmormoy@gmail.com
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Organization name |
FIVI
|
Street address |
Catedrático AgustÃn Escardino
|
City |
Valencia |
ZIP/Postal code |
46980 |
Country |
Spain |
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Platform ID |
GPL10332 |
Series (1) |
GSE46721 |
Transcriptomic effects of miR-30d transient over-expression in human endometrial epithelial cells. |
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