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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.


Related in: MedlinePlus

PCR strategy to amplify GILZ and L-GILZ: (a) structure of GILZ gene, GILZ mRNA and GILZ pseudogene. W and Z represent the primers used in RT-PCR shown in (b) and (d), A and B represent the primers used in the RT-PCR shown in (c), whereas C and D are the primers used in (e). (b) RT-PCR performed with inappropriate primers W–Z. (c) RT-PCR performed with A and B primers. (d) Restriction enzyme digestion performed in the WT PCR product shown in (b). (e) PCR to amplify GILZ. All PCRs used thymus-derived cDNA. The expected lengths of digested fragments are indicated for each amplified gene: (f) gene structure of L-GILZ compared with GILZ. E and F are specific primers to amplify L-GILZ. (g) RT-PCR to amplify L-GILZ in testis.
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f0005: PCR strategy to amplify GILZ and L-GILZ: (a) structure of GILZ gene, GILZ mRNA and GILZ pseudogene. W and Z represent the primers used in RT-PCR shown in (b) and (d), A and B represent the primers used in the RT-PCR shown in (c), whereas C and D are the primers used in (e). (b) RT-PCR performed with inappropriate primers W–Z. (c) RT-PCR performed with A and B primers. (d) Restriction enzyme digestion performed in the WT PCR product shown in (b). (e) PCR to amplify GILZ. All PCRs used thymus-derived cDNA. The expected lengths of digested fragments are indicated for each amplified gene: (f) gene structure of L-GILZ compared with GILZ. E and F are specific primers to amplify L-GILZ. (g) RT-PCR to amplify L-GILZ in testis.

Mentions: When we tried to amplify GILZ by reverse transcriptase PCR (RT-PCR), we found that several couples of primers located along GILZ mRNA (for instance, Forward primer in exon 3-W- and Reverse primer in exon 6-Z) amplified a product even in GILZ knock-out (KO) cells, demonstrating that they amplify other sequences homologous to GILZ (Fig. 1a and b).


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)

PCR strategy to amplify GILZ and L-GILZ: (a) structure of GILZ gene, GILZ mRNA and GILZ pseudogene. W and Z represent the primers used in RT-PCR shown in (b) and (d), A and B represent the primers used in the RT-PCR shown in (c), whereas C and D are the primers used in (e). (b) RT-PCR performed with inappropriate primers W–Z. (c) RT-PCR performed with A and B primers. (d) Restriction enzyme digestion performed in the WT PCR product shown in (b). (e) PCR to amplify GILZ. All PCRs used thymus-derived cDNA. The expected lengths of digested fragments are indicated for each amplified gene: (f) gene structure of L-GILZ compared with GILZ. E and F are specific primers to amplify L-GILZ. (g) RT-PCR to amplify L-GILZ in testis.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0005: PCR strategy to amplify GILZ and L-GILZ: (a) structure of GILZ gene, GILZ mRNA and GILZ pseudogene. W and Z represent the primers used in RT-PCR shown in (b) and (d), A and B represent the primers used in the RT-PCR shown in (c), whereas C and D are the primers used in (e). (b) RT-PCR performed with inappropriate primers W–Z. (c) RT-PCR performed with A and B primers. (d) Restriction enzyme digestion performed in the WT PCR product shown in (b). (e) PCR to amplify GILZ. All PCRs used thymus-derived cDNA. The expected lengths of digested fragments are indicated for each amplified gene: (f) gene structure of L-GILZ compared with GILZ. E and F are specific primers to amplify L-GILZ. (g) RT-PCR to amplify L-GILZ in testis.
Mentions: When we tried to amplify GILZ by reverse transcriptase PCR (RT-PCR), we found that several couples of primers located along GILZ mRNA (for instance, Forward primer in exon 3-W- and Reverse primer in exon 6-Z) amplified a product even in GILZ knock-out (KO) cells, demonstrating that they amplify other sequences homologous to GILZ (Fig. 1a and b).

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.


Related in: MedlinePlus