Multiplex Relative RT-PCR Method for Verification of Differential Gene Expression

Spencer, Weston E.; Christensen, Merrill J.

doi:10.2144/99275rr04

Cited by 55 publications

(40 citation statements)

References 8 publications

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“…For the rat ribosomal protein L19, the primers used were 5Ј-GGA CAG AGT CCA AGG GTC CGC TGC AGTC-3Ј and 5Ј-TCC AAG GGT CCG CTG CAG TC-3Ј. When a co-amplification of NUR77, 20␣-HSD, and L19 message was performed the following primers for 20␣-HSD were used, 5Ј-TGT ATC TCT GAG TTC CCA GG-3Ј and 5Ј-ACT CTT CTA GGG AAG AGC AG-3Ј (15), and the above primer for NUR77 and L19 with a protocol previously described (16). For mice, the 20␣-HSD primers used were 5Ј-TCT TCG GTA CTT TCC TGC TGAT-3Ј and 5Ј-CTG GGG GTG AGT TGC TAAG-3Ј based on the luteal mouse 20␣-HSD sequence (17).…”

Section: Methodsmentioning

confidence: 99%

Prostaglandin F2α-induced Expression of 20α-Hydroxysteroid Dehydrogenase Involves the Transcription Factor NUR77

Stocco¹,

Zhong²,

Sugimoto³

et al. 2000

Journal of Biological Chemistry

120

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Prostaglandin F 2 ␣ (PGF 2 ␣) binding to its receptor on the rat corpus luteum triggers various signal transduction pathways that lead to the activation of a steroidogenic enzyme, 20␣-hydroxysteroid dehydrogenase (20␣-HSD), which in turn catabolizes progesterone. The molecular mechanism underlying PGF 2 ␣-induced 20␣-HSD enzyme activity has not yet been explored. In this report we show, using mice lacking PGF 2 ␣ receptor and pregnant rats, that PGF 2 ␣ is responsible for the rapid and massive expression of the 20␣-HSD gene at the end of pregnancy leading to a decrease in progesterone secretion. We also present evidence that PGF 2 ␣ enhances 20␣-HSD promoter activity. We have determined a region upstream of the ؊1590 position in the 20␣-HSD promoter that confers regulation by PGF 2 ␣ in ovarian primary cells. This region encompasses a unique transcription factor-binding site with a sequence of a NUR77 response element. Deletion of this motif or overexpression of a NUR77 dominant negative protein caused a complete loss of 20␣-HSD promoter activation by PGF 2 ␣. NUR77 also transactivated the 20␣-HSD promoter in transient transfection experiments in corpus luteumderived cells (GG-CL). This induction required the NUR77-transactivating domain. We also show that PGF 2 ␣ induces a very rapid expression of NUR77 that binds to a distal response element located at ؊1599/ ؊1606 but does not interact with another proximal putative NUR77 response element located downstream in the promoter. A rapid increase in NUR77 mRNA was observed in mice corpora lutea just before parturition at a time when 20␣-HSD becomes expressed. This increase in the expression of both genes was not seen in PGF 2 ␣ receptor knockout mice. By using cyclosporin A and PGF 2 ␣ treatment, we established that inhibition of NUR77 DNA binding in vivo prevents PGF 2 ␣ induction of the 20␣-HSD gene in the corpus luteum. Taken together, our results demonstrate, for the first time, that PGF 2 ␣ induces in the corpus luteum the expression of the nuclear orphan receptor and transcription factor, NUR77, which in turn leads to the transcriptional stimulation of 20␣-HSD, triggering the decrease in serum progesterone essential for parturition.In all mammalian species, progesterone plays an essential role in reproduction. The precise timing of both the synthesis and degradation of this steroid hormone is crucial for reproductive success. The expression of enzymes implicated in the synthesis and catabolism of progesterone, therefore, needs to be accurately regulated during the different reproductive states of the animal. In rodents, the corpus luteum, which is the only source of progesterone throughout pregnancy (1), is also able to express the enzyme 20␣-hydroxysteroid dehydrogenase (20␣-HSD)1 that converts progesterone into a biologically inactive steroid, thus playing a key role in the termination of pregnancy and allowing parturition to occur (2). Due to the detrimental effect of 20␣-HSD on luteal progesterone secretion, the corpus luteum of pregnancy does not express...

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Section: Methodsmentioning

confidence: 99%

Prostaglandin F2α-induced Expression of 20α-Hydroxysteroid Dehydrogenase Involves the Transcription Factor NUR77

Stocco¹,

Zhong²,

Sugimoto³

et al. 2000

Journal of Biological Chemistry

120

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“…A 40-l reverse transcription reaction mixture containing hexamers, GSP, or oligo(dT) [12][13][14][15][16][17][18] , 0.9 g of RNA, 0.25 mM dNTP mix, 10 mM dithiothreitol, and 4 units of RNase recombinant inhibitor (Invitrogen) was incubated at 42°C for 2 min when GSP and oligo(dT) [12][13][14][15][16][17][18] were used, or at 25°C for 10 min when random hexamers were used. Superscript II RNase H Ϫ reverse transcriptase (0.2 units) (Invitrogen) was then added, and the mixture was incubated at 42°C for 50 min.…”

Section: Methodsmentioning

confidence: 99%

“…It is known that the various transcripts resulting from alternative splicing can have different lengths of the poly(A) tail (18), and, consequently, some isoforms cannot be detected by using oligo(dT) [12][13][14][15][16][17][18] during the RT experiments. In the present study, after extracting the total RNA from normal thyroid tissues, three different kind of primers were thus used in the first strand cDNA synthesis reaction depending on the experiment, namely random hexamers, GSPs, and oligo(dT) [12][13][14][15][16][17][18] . Random hexamers lead to the production of short cDNA fragments and can therefore be used to avoid secondary structure problems or when the mRNA has a short poly(A) tail.…”

Section: Identification Of the Deletion Variants Of Exon 8 And 14 -Amentioning

confidence: 99%

“…8C) showed that the efficiencies of the PCRs (as given by the slopes of the lines) were the same with both of these species. Under these conditions, the relative difference between the original abundance of these two samples was taken to be 2 n , where n is the difference between the number of cycles necessary to reach a threshold value (12) (Fig. 8B).…”

Section: Identification Of the Deletion Variants Of Exon 8 And 14 -Amentioning

confidence: 99%

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Increasing Diversity of Human Thyroperoxidase Generated by Alternative Splicing

Ferrand

Fourn²,

Franc

2003

Journal of Biological Chemistry

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The human thyroperoxidase (hTPO) gene is composed of 17 exons. The longest complete cDNA sequence determined so far contains a full-length hTPO (TPO1) encoding a 933-amino acid polypeptide. Several mRNA species encoding for hTPO isoforms are present in normal thyroid tissues, including TPO2 with exon 10 deleted and TPOzanelli with exon 16 deleted. In the present study, we established the existence of two new single-spliced transcripts, TPO4 and TPO5, lacking exons 14 and 8, respectively. Upon transfecting the TPO4 cDNA into Chinese hamster ovary cells, it was observed that TPO4 is able to reach the cell surface, is enzymatically active, and is able to be recognized by a panel of 12 monoclonal antibodies directed against hTPO, whereas TPO5 does not fold correctly and is unable to reach the cell surface. In normal tissues, the expression of TPO4 mRNA was examined by performing quantitative reverse transcription PCR. This deleted TPO mRNA amounted to 32 ؎ 11% of the total TPO mRNAs. In the same tissues, the TPO2, TPOzanelli, and TPO5 amounted to 35 ؎ 12%, 36 ؎ 14%, and ϳ10%, respectively. The sum of these four species (not including TPO1) was more than 100%, possibly due to the presence of multispliced mRNAs. This possibility was tested, and three new variants were identified: TPO2/3, lacking exons 10 and 16, TPO2/4, lacking exons 10 and 14, and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16. In conclusion, these results indicate the existence of five new transcripts. One of them, TPO4, codes for an enzymatically active protein, whereas TPO5 is unable to fold correctly. The functional significance of the other newly spliced mRNA variants still remains to be elucidated, but these results might help to explain the heterogeneity of the hTPO purified from the thyroid gland. Thyroperoxidase (TPO)1 is the key enzyme in the process of thyroid hormone synthesis. The human TPO gene is about 150 kbp in size, is located on chromosome 2, locus 2p25, and consists of 17 exons and 16 introns (for a review, see Ref. 1). The complete sequence of the human TPO coding region is known (2-4). The full-length 3048-bp transcript (TPO1) codes for a protein consisting of 933 amino acids, which have a large extracellular domain, a transmembrane domain consisting of 60 residues, and a short intracytoplasmic tail consisting of 60 residues. Two other transcripts have been described, namely TPO2, in which exon 10 is spliced out, and TPOzanelli (TPO3), in which exon 16 is spliced out. TPO2 and TPO3 have been found to occur in normal thyroid tissues as well as in Graves' tissues (2, 5, 6). These two forms code for proteins consisting of 876 and 929 residues, respectively. TPO2 is rapidly degraded after its synthesis, does not reach the cell surface, and does not have any enzymatic activity (7), whereas TPO3 is able to reach the cell surface and shows enzymatic activity (8).After being purified from the human thyroid gland, TPO is known to show up in SDS-PAGE under reducing conditions as a double band of...

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“…For SM-PCR we followed the protocol of Spencer and Christensen (Spencer and Christensen, 1999) with minor modifications. Essentially, the cDNA species of interest was co-amplified with β-actin cDNA (see Table 1 for primer sequences) over a range of cycles, followed by 2% agarose electrophoresis and ethidium bromide stain.…”

Section: Semi-quantitative 'Multiplex' Polymerase-chain Reaction (Sm-mentioning

confidence: 99%

PTHrP [67-86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression

Luparello

Sirchia

Pupello

2003

Journal of Cell Science

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It was previously reported that a midregion domain of parathyroid hormone-related protein (PTHrP), that is, [67-86]-amide, is able to restrain growth and promote matrigel penetration by the 8701-BC cell line, derived from a biopsy fragment of a primary ductal infiltrating carcinoma of the human breast, and that cell invasion in vitro is drastically impaired by inactivation of urokinase-plasminogen activator (uPa). In this study we started a more detailed investigation of the possible effects on gene expression arising from the interaction between PTHrP [67-86]-amide and 8701-BC breast cancer cells by a combination of conventional-, differential display-and semi-quantitative multiplex-polymerase chain reaction (PCR)assays. We present here the first evidence that the upregulation of some stress-related genes, most noticeably heat shock factor binding protein-1(hsbp1) and heat shock protein 90 (hsp-90), is involved in the acquisition of an in vitro more invasive phenotype by cells treated with midregion PTHrP. This is conceivably accomplished by sequestering and inactivating heat shock factor-1 (hsf1) which is able to recognize Ets transcription-factor-binding sites present in some gene promoters, such as those of uPa and matrix metalloprotease-1 (MMP-1). In fact,our data show that incubation of PTHrP [67-86]-amide-treated cells with either antisense hsbp1-oligonucleotide or geldanamycin, an hsp90-inactivating antibiotic, results in downregulation of uPa and upregulation of MMP-1, and in a prominent inhibition of cell invasion in matrigel-containing Transwell chambers. Alternatively, incubation of untreated 8701-BC cells with quercetin, a flavonoid known to decrease the amount of free hsf1, is found to induce upregulation of uPa and downregulation of MMP-1, and an increase of matrigel invasion by cells, thus providing further supporting data of the involvement of hsf unavailability on the modulation of uPa and MMP-1 expression and on cell invasive behaviour. These studies confirm a previous postulate that over-secretion of uPa, rather than of other extracellular proteases, is a primary condition for the increase of invasive activity triggered by PTHrP [67-86]-amide in vitro,and support a role for midregion forms of PTHrP in potentially affecting pathological mammary growth and differentiation. They also identify two new key protagonists in the complex scenario of breast tumor cell invasiveness in vitro, that is, hsbp1 and hsp90, which deserve further and more extensive studies as potential and attractive molecular targets for anti-breast cancer treatments.

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Multiplex Relative RT-PCR Method for Verification of Differential Gene Expression

Cited by 55 publications

References 8 publications

Prostaglandin F2α-induced Expression of 20α-Hydroxysteroid Dehydrogenase Involves the Transcription Factor NUR77

Prostaglandin F2α-induced Expression of 20α-Hydroxysteroid Dehydrogenase Involves the Transcription Factor NUR77

Increasing Diversity of Human Thyroperoxidase Generated by Alternative Splicing

PTHrP [67-86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression

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