microRNAs (miRNAs) are small, functional, non‐coding RNAs. miRNAs are transcribed as long primary transcripts (primary precursors) that are processed to the ∼75 nt precursors (pre‐miRNAs) by the ...nuclear enzyme Drosha. The ∼22 nt mature miRNA is processed from the pre‐miRNA by the RNase III Dicer. The vast majority of published studies to date have used northern blotting to detect the expression of miRNAs. We describe here a sensitive, high throughput, real‐time PCR assay to monitor the expression of miRNA precursors. Gene‐specific primers and reverse transcriptase were used to convert the primary precursors and pre‐miRNAs to cDNA. The expression of 23 miRNA precursors in six human cancer cell lines was assayed using the PCR assay. The miRNA precursors accumulated to different levels when compared with each other or when a single precursor is compared in the various cell lines. The precursor expression profile of three miRNAs determined by the PCR assay was identical to the mature miRNA expression profile determined by northern blotting. We propose that the PCR assay may be scaled up to include all of the 150+ known human miRNA genes and can easily be adaptable to other organisms such as plants, Caenorhabditis elegans and Drosophila.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input ...copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts ...is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named 'hypoxia-induced noncoding ultraconserved transcripts' (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.
Four quantitative reverse transcription–PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human β-globin ...open reading frame/c-myc 3′-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to β-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r2 obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.
Our previous study described a real-time PCR method to quantify microRNA (miRNA) precursors using SYBR green detection T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, ...e43. The present study adapted the assay to a 384-well format and expanded it to include primers to 222 human miRNA precursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression in many commonly used human cancer cell lines.
Down syndrome (DS), or Trisomy 21, is the most common genetic cause of cognitive impairment and congenital heart defects in the human population. To date, the contribution of microRNAs (miRNAs) in DS ...has not been investigated. Bioinformatic analyses demonstrate that human chromosome 21 (Hsa21) harbors five miRNA genes; miR-99a, let-7c, miR-125b-2, miR-155, and miR-802. MiRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments demonstrate that these miRNAs are overexpressed in fetal brain and heart specimens from individuals with DS when compared with age- and sex-matched controls. We hypothesize that trisomic 21 gene dosage overexpression of Hsa21-derived miRNAs results in the decreased expression of specific target proteins and contribute, in part, to features of the neuronal and cardiac DS phenotype. Importantly, Hsa21-derived miRNAs may provide novel therapeutic targets in the treatment of individuals with DS.
Benign metastasizing leiomyomas are rare tumors, which are typically found in the lungs and, thus, might be confused with leiomyosarcomas. Further, it is not clear whether the term "benign ...metastasizing leiomyoma" is a misnomer and whether these lesions actually represent low-grade malignant tumors that have a low proliferation index. Micro-RNAs (miRNAs) are small noncoding RNAs, which repress translation. The altered expression of miRNAs has been strongly correlated with the malignant phenotype. In this study, the histologic features, Ki67 index, p53, bcl-2, and miRNA expression were studied in 15 leiomyosarcomas (11 primary lesions and 4 metastases), 8 leiomyomas, and 10 cases of benign metastasizing leiomyoma (9 pulmonary lesions and 1 primary uterine lesion). As expected, the Ki67 index for the benign metastasizing leiomyomas was equivalent to that for the leiomyomas and statistically less than that for the leiomyosarcomas. The mean index was 2.3% (range: 0.9% to 8.8%) for the leiomyomas and 3.4% (range: 0.7% to 8.1%) for the benign metastasizing leiomyomas compared with 28.6% (range: 14.4% to 62.0%) for the leiomyosarcomas (P<0.025). The miRNA, miR-221, which has been associated with a variety of cancers, was detected by in situ hybridization in 13/15 leiomyosarcomas, 0/8 leiomyomas, and 0/10 benign metastasizing leiomyomas. In conclusion, benign metastasizing leiomyomas are indeed most likely benign lesions, and up-regulation of miR-221 expression is an accurate way to differentiate leiomyosarcoma from benign metastasizing leiomyoma.
Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of ...initial cervical HPV infection, showed that miRNAs let-7c, -99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis.
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