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| Status |
Public on Jul 26, 2018 |
| Title |
Neonatal Clone 3_conventionalRT-PCR_ground_control |
| Sample type |
RNA |
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| Source name |
Primary cardiac tissue
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Cardiovascular progenitor cell
culture condition: Biocell (BioServe Space Technologies) - Ground
duration: 12 days
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| Growth protocol |
CPCs were flown to the ISS where they were cultured for 12 days and fixed in RNA protect. Clone-, patient-, and passage-matched controls were seeded and grown on the ground for 12 days as a control.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy Mini Kit or using TRIzol (for microRNA) and alcohol-based precipitation
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| Label |
SYBR Green
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| Label protocol |
mRNA: PCR assays for genes were performed using individual primer pairs (ID_REF A01-B04), cDNA prepared as described, and SYBR Green Master Mix (Promega, Madison, WI). Reverse transcription was performed from 2000 ng total RNA using the Superscript III kit (Life Technologies, Carlsbad, CA). Quantitative real-time PCR were performed (iQ5 thermal cycler, BioRad) with 45 cycles at 95 oC for 15 seconds, 57 oC for 60 seconds, and 72 oC for 30 seconds (with the exception of YAP1, which was run at 45 cycles at 95 oC for 15 seconds, 55 oC for 60 seconds, and 72 oC for 30 seconds).
microRNA: PCR assays for microRNA gene expression were performed using individual primer pairs (ID_REF B05-B08) , cDNA prepared as described, and QuantiText SYBR Green PCR Master Mix (Qiagen, Valencia, CA). Reverse transcription was performed from 500 ng total RNA using the miScript SYBR Green PCR master mix and 10X universal primer mix (Qiagen, Valencia, CA). Quantitative real-time PCR were performed (iQ5 thermal cycler, BioRad) with 40 cycles at 94 oC for 15 seconds, 55 oC for 30 secons, and 70 oC for 30 seconds.
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| Hybridization protocol |
n/a
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| Scan protocol |
n/a
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| Description |
Ground control
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| Data processing |
Normalization and comparative Ct method analysis per clone was performed using Qiagen's web-based software package: http://www.qiagen.com/ us/shop/genes-and-pathways/data-analysis-center-overview-page/
For normalization, we used the average of GAPDH and ACTB.
Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)].
Fold changes per clone were exported to Prism for subsequent analysis. Fold Change worksheet reports test/control (i.e., ISS-cultured CPCs/ground CPCs) ratios.
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| Submission date |
Feb 13, 2018 |
| Last update date |
Jul 26, 2018 |
| Contact name |
Jonathan Baio |
| E-mail(s) |
jbaio@llu.edu
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| Organization name |
Loma Linda University School of Medicine
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| Department |
Pathology and Human Anatomy
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| Lab |
Kearns-Jonker
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| Street address |
24941 Stewart St, Room 326
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| City |
Loma Linda |
| State/province |
CA |
| ZIP/Postal code |
92354 |
| Country |
USA |
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| Platform ID |
GPL24605 |
| Series (2) |
| GSE110562 |
Real-time quantitative PCR analysis of human cardiovascular progenitor cells flown aboard the International Space Station [conventional RT-PCR] |
| GSE110563 |
Real-time quantitative PCR analysis of human cardiovascular progenitor cells flown aboard the International Space Station |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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