|
| Status |
Public on Jul 14, 2015 |
| Title |
CX1 T21 |
| Sample type |
RNA |
| |
|
| Source name |
Bone Marrow MSCs
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Bone Marrow MSCs
differentiation: day 21 of osteogenic differentiation
group: Coxarthrosis
|
| Treatment protocol |
Osteogenic differentiation cultures were performed in 24-well tissue culture plates at a seeding density of (30,000 cells/cm2) using commercial culture media with supplements according to manufacturer instructions (Promocell Cat# C-28013)
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| Growth protocol |
MSCs were obtained from bone marrow aspirates and after isolation by plastric adhaerence and expansion MSCs were characterized by flow cytometry and lineage multipotential Phenotypic characterization of isolated MSCs was performed by flow citometry in a Gallios flow cytometer (Beckman Coulter). Cells recovered at the third passage were washed with PBS prior incubation during 30 minutes at 4ºC with specific phycoerythrin conjugated antibodies and their isotype matched antibodies (all from Miltenyi Biotech). Expression of CD105, CD73 and CD90, and lack of expression of CD45, CD34, and CD14 were the minimal criteria required prior to evaluation of their differentiation potential towards chondrogenic, adipogenic and osteogenic lineages, assessed by histochemical stainings.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For miRNA RT-PCR, total RNA was isolated with miRCURY isolation kit (Exiqon) and cDNA Synthesis was carried out using 10 ng of RNA and the Universal cDNA synthesis kit II (Exiqon). Following reverse transcription, cDNAs were 1:100 diluted and loaded onto the real time PCR plates using a 1:1 mixture of Exilent SYBR Green master mix 2x (Exiqon). Real Time PCR reactions were performed in a 7900HT Fast Real time PCR System (Applied Biosystems) under the following conditions: 95˚C 10min, 45 cycles of 95 ˚C 10s and 60 ˚C 1min.
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| Label |
SYBR® Green
|
| Label protocol |
n/a
|
| |
|
| Hybridization protocol |
n/a
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| Scan protocol |
n/a
|
| Description |
MSCs at day 21 of osteogenic differentiation
SAMPLE 3
|
| Data processing |
After run completion, Ct values were calculated using the SDS 2.4 software (Applied Biosystems) and data were analyzed using the StatMiner® software (Integromics)
Start Activity 1 file(s) uploaded This is the number of files successfully uploaded in this project.
Start Activity Undetermined values replaced by 40.00 This is the value used for replacing undetermined Ct values.
Start Activity Number of experimental factors: 4 Experiment design has one or more experimental factors columns added.
Start Activity 1 plate(s) uploaded This is the number of plates successfully uploaded in this project.
Start Activity 24 sample(s) uploaded This is the number of unique samples successfully uploaded in this project.
Start Activity 46 detector(s) uploaded This is the number of unique detectors from the different samples successfully uploaded in this project.
Start Activity 2304 measurement(s) uploaded This is the number of wells with a valid Ct successfully uploaded in this project.
Start Activity 384 well(s) uploaded This is the number of wells successfully uploaded in this project.
Start Activity Type of experiment: Real-Time qPCR Unless you are using AB miRNA assay version 1.0 cards, 'Type of experiment' should be set as 'Real-Time qPCR'.
Start Activity 162 undetermined well(s) (replaced by Ct 40) Wells with Undetermined Ct are replaced by a high Ct value that can be filter out as not detected during the analysis.
Start Activity 0 empty well(s) Wells with empty Ct found. RealTime StatMiner® will try to impute empty Ct using the biological replicates.
Start Activity Main experimental factor: NewExperimentFactor1.1. One of the experimental factors (or a combination of them) must be selected to perform differential expression studies.
Start Exception Experimental factor name NewExperimentFactor1(1) is R-incompatible (renamed as NewExperimentFactor1.1.) The label of the experimental factor has incompatible characters that will be automaticall renamed.
Qualify Activity 3 well(s) flagged as outlier(s) using Grubbs test Technical replicates were flagged and automatically removed.
Qualify Exception Sample CX18.T0 flagged as outlier (6.78 flags) using MAD test This sample is poorly correlated with its biological replicates. Select biological replicates automatic removal from Project Options tool to automatically discard it.
Qualify Exception Sample CX17.T10 flagged as outlier (7.51 flags) using MAD test This sample is poorly correlated with its biological replicates. Select biological replicates automatic removal from Project Options tool to automatically discard it.
Qualify Exception Sample CX18.T21 flagged as outlier (5.75 flags) using MAD test This sample is poorly correlated with its biological replicates. Select biological replicates automatic removal from Project Options tool to automatically discard it.
Qualify Exception Sample FS15.T0 flagged as outlier (3.48 flags) using MAD test This sample is poorly correlated with its biological replicates. Select biological replicates automatic removal from Project Options tool to automatically discard it.
Filter Activity 2 detector(s) have a Ct value equal to or higher than 38 in at least 3 samples in group control.T0 Detectors with high Ct values were flagged as not amplified.
Filter Activity 1 detector(s) have a Ct value equal to or higher than 38 in at least 3 samples in group control.T10 Detectors with high Ct values were flagged as not amplified.
Filter Activity 3 detector(s) have a Ct value equal to or higher than 38 in at least 3 samples in group control.T21 Detectors with high Ct values were flagged as not amplified.
Filter Activity 3 detector(s) have a Ct value equal to or higher than 38 in at least 4 samples in group case.T0 Detectors with high Ct values were flagged as not amplified.
Filter Activity 2 detector(s) have a Ct value equal to or higher than 38 in at least 4 samples in group case.T10 Detectors with high Ct values were flagged as not amplified.
Filter Activity 3 detector(s) have a Ct value equal to or higher than 38 in at least 4 samples in group case.T21 Detectors with high Ct values were flagged as not amplified.
Normalize Activity 6 candidate endogenous control(s) analyzed using Genorm RealTime StatMiner® has automatically evaluated the optimal endogenous controls.
Normalize Activity Project normalized using hsa-miR-103a-3p, hsa-miR-191-5p, hsa-miR-30c-5p, SNORD49A This is the list of endogenous controls selected to normalize the Ct values.
Differential expression Activity Differential expression using the Parametric test (Limma) This statistical test was used in the differential expression study.
Differential expression Activity Benjamini-Hochberg False Discovery Rate p-value adjustment This method corrected the p-value used in the differential expression study statistical test
Differential expression Activity Adjusted p-value cut-off 0.05 P-value cut-off for statistically significant results was set.
Differential expression Activity Differential expression of case.T0 using control.T0 as background This pair of samples was used in the differential expression experiment.
Differential expression Activity Differential expression of case.T10 using control.T10 as background This pair of samples was used in the differential expression experiment.
Differential expression Activity Differential expression of case.T21 using control.T21 as background This pair of samples was used in the differential expression experiment.
Differential expression Activity Differential expression of case.T10,case.T21 using case.T0 as background This pair of samples was used in the differential expression experiment.
Differential expression Activity Differential expression of case.T21 using control.T10 as background This pair of samples was used in the differential expression experiment.
Differential expression Activity Differential expression of case.T21 using case.T10 as background This pair of samples was used in the differential expression experiment.
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| |
|
| Submission date |
Jul 13, 2015 |
| Last update date |
Jul 14, 2015 |
| Contact name |
José Ramón Lamas Lamas |
| E-mail(s) |
jrlamas@gmail.com
|
| Phone |
+34913303615
|
| Organization name |
Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC)
|
| Department |
Rheumatology
|
| Lab |
Medicina y Cirugía Experimental. Laboratorio 2
|
| Street address |
Calle Profesor Martín Lagos s/n. Hospital Clínico san Carlos
|
| City |
Madrid |
| State/province |
MADRID |
| ZIP/Postal code |
28220 |
| Country |
Spain |
| |
|
| Platform ID |
GPL20685 |
| Series (1) |
| GSE70856 |
Real-time quantitative PCR analysis for validation of miRNAs expressed during osteogenic differentiation of bone marrow MSCs versus healthy donors MSCs |
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