|
| Status |
Public on Jun 09, 2010 |
| Title |
HKU_Liver_0175_AN_RS-260918_22185_S4_UPDATED_MIRNA |
| Sample type |
RNA |
| |
|
| Source name |
liver
|
| Organism |
Homo sapiens |
| Characteristics |
individual: 175
tissue: adjacent liver non-tumor
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples are processed in parallel in 96-well plates to minimize potential variation. Reaction purification is achieved using RLT/BME homogenization with Trizol vortex, followed by chloroform extraction and isopropanol precipitation.
|
| Label |
SYBR green
|
| Label protocol |
When SYBR green binds to duplex DNA, its fluorescence is increased. SYBR green fluorescence is monitored during amplification by the 7900 HT PCR instrument (Ambion Inc.), generating amplification curves for quantitation.
|
| |
|
| Hybridization protocol |
Following reverse transcription, quadruplicate measurements of 2 uL of cDNA were made in 10 uL final reaction volumes by qPCR in a 384-well optical PCR plate using a 7900 HT PCR instrument (Applied Biosystems). SYBR green PCR mix contained 5 uL of 2x SYBR green PCR master mix (Applied Biosystems), 1.4 uL of water, 0.8 uL of 10 mM universal primer, 0.8 uL of 10 uM LNA-Rprimer, and 2 uL of sample. qPCR was performed using the manufacturer’s recommended conditions.
|
| Scan protocol |
SYBR green fluorescence is monitored during amplification by the 7900 HT PCR instrument (Ambion Inc.), generating amplification curves for quantitation. Dissociation curves were typically generated post-run for analysis of amplicon species.
|
| Description |
Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide, reflecting the need for a better understanding of hepatocarcinogenesis. In this study, we examined the genome-wide expression profiles of both miRNAs and mRNAs from paired tumor and adjacent non-tumor tissues from a cohort of 96 HCC patients in Hong Kong where hepatitis B virus (HBV) is endemic. The comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that it is an important regulator for normal mitochondrial metabolism. A decreased level of miR-122 leads to an increase in expression of miR-122 seed-matched genes, a loss of mitochondrial metabolic function, and a decrease in the expression of many miR-122 secondary targets. These secondary targets are prognostic markers for HCC patients. Transcriptome profiling data from an additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. To test the HCC findings in normal mouse liver, we used an antagomir against miR-122 and identified altered gene expression profiling by microarray analysis in mouse in vivo experiments. The mouse data recapitulated the human HCC findings. Our anti-miR122 data further provided a direct link between increases in the mRNA levels of miR-122 controlled genes and impairments of mitochondrial metabolism. In summary, we find miR-122 loss may be detrimental to mitochondrial-related metabolisms in sustaining critical liver function and contribute to the morbidity and mortality of liver cancer patients.
|
| Data processing |
Data were loaded into the Rosetta Resolver system (Rosetta Biosoftware, Seattle, WA) and processed using the qPCR algorithm. Intensities were calculated by setting a threshold within the linear range of the amplification curve and comparing the cycle time at which the experimental sample crossed the threshold to a standard curve. Standard curve dilutions of synthetic oligonucleotides ranging from 10 nM to 10 fM were performed in TE that contained 100 ng/mL of total yeast RNA (Ambion, Inc.).
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| |
|
| Submission date |
May 28, 2010 |
| Last update date |
Jun 08, 2010 |
| Contact name |
Julja Burchard |
| Organization name |
Merck & Co., Inc.
|
| Department |
Computational & Systems Biology
|
| Street address |
33 Avenue Louis Pasteur
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02176 |
| Country |
USA |
| |
|
| Platform ID |
GPL10457 |
| Series (1) |
| GSE22058 |
miR-122 as a Regulator of Mitochondrial Metabolic Gene Network in Hepatocellular Carcinoma |
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