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A focused Real Time PCR strategy to determine GILZ expression in mouse tissues.

Cari L, Ricci E, Gentili M, Petrillo MG, Ayroldi E, Ronchetti S, Nocentini G, Riccardi C - Results Immunol (2015)

Bottom Line: Glucocorticoid-Induced Leucine Zipper (GILZ) is a glucocorticoid-inducible gene that mediates glucocorticoid anti-inflammatory effects.An inappropriate primer pair amplified also GILZ pseudogene and TSC22d1iso3, thus producing misleading results.This quantitative evaluation may be used to better characterize the role of GILZ and L-GILZ in mice and may be translated to humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Pharmacology, University of Perugia Medical School, P.le L. Severi 1, 06132 Perugia, Italy.

ABSTRACT
Glucocorticoid-Induced Leucine Zipper (GILZ) is a glucocorticoid-inducible gene that mediates glucocorticoid anti-inflammatory effects. GILZ and the isoform L-GILZ are expressed in a variety of cell types, especially of hematopoietic origin, including macrophages, lymphocytes and epithelial cells, and strongly upregulated upon glucocorticoid treatment. A quantitative analysis of GILZ expression in mouse tissues is technically difficult to perform because of the presence of a pseudogene and the high homology of GILZ gene with other genes of TSC22 family. We here propose specific primer pairs to be used in Real Time PCR to avoid unwanted amplification of GILZ pseudogene and TSC-22 family member d1iso3. These primer pairs were used to determine GILZ and L-GILZ expression, in either untreated or in vivo and in vitro dexamethasone-treated tissues. Results indicate that GILZ and L-GILZ are upregulated by glucocorticoids, being GILZ more sensitive to glucocorticoid induction than L-GILZ, but they are differently expressed in all examined tissues, confirming a different role in specific cells. An inappropriate primer pair amplified also GILZ pseudogene and TSC22d1iso3, thus producing misleading results. This quantitative evaluation may be used to better characterize the role of GILZ and L-GILZ in mice and may be translated to humans.

No MeSH data available.


Modulation of GILZ and L-GILZ investigated by Real Time PCR in fresh mouse tissues 3 h after in vivo DEX treatment (10 mg/kg). The fold values in each group were calculated versus the same tissues from untreated mice, set equal to 1, on a base 2 log scale. Results are mean of triplicates±SD of two experiments.
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f0015: Modulation of GILZ and L-GILZ investigated by Real Time PCR in fresh mouse tissues 3 h after in vivo DEX treatment (10 mg/kg). The fold values in each group were calculated versus the same tissues from untreated mice, set equal to 1, on a base 2 log scale. Results are mean of triplicates±SD of two experiments.

Mentions: Because GILZ is a GC-induced gene, we treated mice with the synthetic glucocorticoid dexamethasone (DEX) for 3 h, and recovered tissues from diverse organs. Data in Fig. 3 clearly show how both GILZ (Fig. 3a) and L-GILZ (Fig. 3b) are upregulated after GC exposure in almost all tissues. GILZ is more sensitive to GC induction than L-GILZ, reaching high levels of expression especially in thymus and in lung.


A focused Real Time PCR strategy to determine GILZ expression in mouse tissues.

Cari L, Ricci E, Gentili M, Petrillo MG, Ayroldi E, Ronchetti S, Nocentini G, Riccardi C - Results Immunol (2015)

Modulation of GILZ and L-GILZ investigated by Real Time PCR in fresh mouse tissues 3 h after in vivo DEX treatment (10 mg/kg). The fold values in each group were calculated versus the same tissues from untreated mice, set equal to 1, on a base 2 log scale. Results are mean of triplicates±SD of two experiments.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664734&req=5

f0015: Modulation of GILZ and L-GILZ investigated by Real Time PCR in fresh mouse tissues 3 h after in vivo DEX treatment (10 mg/kg). The fold values in each group were calculated versus the same tissues from untreated mice, set equal to 1, on a base 2 log scale. Results are mean of triplicates±SD of two experiments.
Mentions: Because GILZ is a GC-induced gene, we treated mice with the synthetic glucocorticoid dexamethasone (DEX) for 3 h, and recovered tissues from diverse organs. Data in Fig. 3 clearly show how both GILZ (Fig. 3a) and L-GILZ (Fig. 3b) are upregulated after GC exposure in almost all tissues. GILZ is more sensitive to GC induction than L-GILZ, reaching high levels of expression especially in thymus and in lung.

Bottom Line: Glucocorticoid-Induced Leucine Zipper (GILZ) is a glucocorticoid-inducible gene that mediates glucocorticoid anti-inflammatory effects.An inappropriate primer pair amplified also GILZ pseudogene and TSC22d1iso3, thus producing misleading results.This quantitative evaluation may be used to better characterize the role of GILZ and L-GILZ in mice and may be translated to humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Pharmacology, University of Perugia Medical School, P.le L. Severi 1, 06132 Perugia, Italy.

ABSTRACT
Glucocorticoid-Induced Leucine Zipper (GILZ) is a glucocorticoid-inducible gene that mediates glucocorticoid anti-inflammatory effects. GILZ and the isoform L-GILZ are expressed in a variety of cell types, especially of hematopoietic origin, including macrophages, lymphocytes and epithelial cells, and strongly upregulated upon glucocorticoid treatment. A quantitative analysis of GILZ expression in mouse tissues is technically difficult to perform because of the presence of a pseudogene and the high homology of GILZ gene with other genes of TSC22 family. We here propose specific primer pairs to be used in Real Time PCR to avoid unwanted amplification of GILZ pseudogene and TSC-22 family member d1iso3. These primer pairs were used to determine GILZ and L-GILZ expression, in either untreated or in vivo and in vitro dexamethasone-treated tissues. Results indicate that GILZ and L-GILZ are upregulated by glucocorticoids, being GILZ more sensitive to glucocorticoid induction than L-GILZ, but they are differently expressed in all examined tissues, confirming a different role in specific cells. An inappropriate primer pair amplified also GILZ pseudogene and TSC22d1iso3, thus producing misleading results. This quantitative evaluation may be used to better characterize the role of GILZ and L-GILZ in mice and may be translated to humans.

No MeSH data available.