http://www.gene-quantification.net/livak-2001.pdf

Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method

Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.

/pdf/analyzing-real-time-pcr-data-by-the-comparative-c-t-method-tnacbujy1o.pdf

Analyzing real-time PCR data by the comparative C(T) method.

Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.

/pdf/accurate-normalization-of-real-time-quantitative-rt-pcr-data-34rwzsvnu5.pdf

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes

Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments.

Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.

Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

/pdf/the-miqe-guidelines-minimum-information-for-publication-of-27m8n04lrd.pdf

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

MicroRNA (miRNA ) alterations are involved in the initiation and progression of human cancer. The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target protein- coding genes involved in cancer.

MicroRNA signatures in human cancers

(unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Gastrointestinal Cancer: Targets and Therapy 2014:4 75–87 Gastrointestinal Cancer: Targets and Therapy Dovepress

Pathology, genetic alterations, and targets of differentially expressed microRNAs in pancreatic cancer

In Situ Detection of MicroRNAs in Paraffin-Embedded, Formalin-Fixed Tissues: Different Methodologies and Co-localization with Possible Targets

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC

Breast cancer is the most commonly diagnosed cancer and the second leading cause of death among women. Triple Negative Breast Cancer (TNBC) represents 10-20% of breast cancer patients. It is considered the most aggressive and challenging type of breast cancer due to the lack of effective targeted therapies. Metastatic TNBC commonly migrates to the lung, brain and liver. Chemotherapy is the only option for these patients and it is hard to predict the success of chemotherapy. It is crucial to investigate the molecular changes leading to TNBC and develop new therapies targeting the molecular changes that cause this disease. microRNA (miRNA) has been implicated in many cancers by deregulating the expression of genes having key roles in cancer initiation and/or progression. We measured the mature levels of miR-205 in five breast cell lines: MCF10A non-tumorigenic normal like epithelial cell line, MCF7 cancer cell line expressing both estrogen and progesterone receptors (ER+/PR+); MDA-MB-436, MDA-MB-231 and BT549 TNBC cell lines (ER-/PR-/HER2-). We also assayed miR-205 in 33 human breast tissues (benign, tumor and metastatic). miR-205 levels were significantly down regulated in breast cancer cell lines compared to normal-like breast epithelial cells and in tumor and metastatic tissues compared to benign. We identified two miR-205 predicted binding sites in the 3’ untranslated region of the high mobility group gene, HMGB3. Both Dual-luciferase reporter assay and Western blotting confirmed that miR-205 regulates HMGB3. To explore the function of miR-205 and HMGB3 in breast cancer, WST-1 proliferation and Matrigel invasion assays were done using the TNBC cell lines MDA-MB-231 and BT549 transiently transfected with precursor miR-205 oligonucleotide (pre-miR-205 oligo) or HMGB3 small interfering RNA (siRNA). Both treatments reduced the proliferation and invasion of the cancer cells, demonstrating that knockdown of HMGB3 with miRNA or siRNA is effective in reducing the malignant phenotype of this protein. The messenger RNA (mRNA) levels of HMGB3 significantly increased in the tumor and metastatic groups in a stage dependent manner suggesting a tumor initiating and progression role. Western blotting of HMGB3 protein from 5 matched pairs of breast tissue showed that HMGB3 protein was not detected in benign tissues but was present in tumor. In conclusion, regulation of HMGB3 by miR-205 reduced both proliferation and invasion of breast cancer cells. Since the down regulation of miR-205 in patients’ tumor specimens correlate with the high HMGB3 protein levels in tumor, our findings suggest that reintroducing miR-205 and targeting HMGB3 are potential therapies for TNBC.

Citation Format: Ola Elgamal, Jong-Kook Park, Thomas D. Schmittgen. miR-205 functions as a tumor suppressor in breast cancer by targeting HMGB3. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4204. doi:10.1158/1538-7445.AM2013-4204

Abstract 4204: miR-205 functions as a tumor suppressor in breast cancer by targeting HMGB3.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC

Pancreatic ductal adenocarcinoma is believed to arise from exocrine acinar cells following trans-differentiation to cells with a more epithelial phenotype. A pancreas enriched cluster of miRNAs (miR-216a/b-217) is located on chromosomes 2 and 11 in humans and mice, respectively. These miRNAs are significantly reduced in patient's tumor specimens and during tumor progression in a transgenic mouse model of pancreatic ductal adenocarcinoma. To investigate a role for miR-216/-217 during acinar ductal trans-differentiation, we used a model by which primary mouse pancreatic acini trans-differentiate into epithelial-like cells when cultured on matrigel. Over a 4 day culture period, the acini develop an epithelial morphology and display reduced mRNA expression of the acinar marker amylase, increased expression of the epithelial marker cytokeratin-19 (KRT19) and reduced expression miR-216/-217. Forced expression of miR-217 using a lentiviral vector (LV) decreased KRT19 mRNA expression compared to the empty vector control treated cells on day 4 of the trans-differentiation. To implicate miR-217 targets in regulating acinar trans-differentiation, we considered REST/NRSF, a transcriptional repressor of neuronal genes in non-neuronal tissues. REST mRNA expression increased during the 4 day trans-differentiation implying regulation of REST by miR-217. Regulation of REST by miR-217 was validated using a luciferase assay and western blotting. Furthermore, two REST target genes were upregulated by the miR-217 LV on day 4 of the trans-differentiation. Studies are ongoing to determine if this increase in expression results from miR-217 suppressing REST protein levels. Additionally, miR-217 LV infection of 3 pancreas cancer cell lines reduced REST protein and caused significant cell death. Our current focus is to establish a link between miR-217 suppression of REST and cell death. We report a connection between reduced miR-217 in several pancreas model systems. We hypothesize a novel role for REST in pancreas homeostasis and cancer.

Citation Format: Ana Clara P. Azevedo-Pouly, Jinmai Jiang, Dhruvitkumar Sutaria, Thomas Schmittgen. Regulation of NRSF/REST by miR-217; Implications during pancreatic acinar ductal trans-differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4187. doi:10.1158/1538-7445.AM2013-4187

Thomas D. Schmittgen

Papers

Pathology, genetic alterations, and targets of differentially expressed microRNAs in pancreatic cancer

In Situ Detection of MicroRNAs in Paraffin-Embedded, Formalin-Fixed Tissues: Different Methodologies and Co-localization with Possible Targets

Abstract 4204: miR-205 functions as a tumor suppressor in breast cancer by targeting HMGB3.

Abstract 4187: Regulation of NRSF/REST by miR-217; Implications during pancreatic acinar ductal trans-differentiation.

Dual Epigenetic Control of CCAAT/Enhancer Binding Protein α (C/EBPα) Expression in Acute Myeloid Leukemia.