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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.


Real Time PCR quantification in fresh mouse tissues: (a) and (b) GILZ and L-GILZ expression was normalized using the housekeeping gene GAPDH. All values were calculated with respect of thymus, set equal to 1 and expressed in a base 2 log scale. (c) Comparison of GILZ and L-GILZ in the same tissues as in (a) and (b) in which values were considered relatively to the housekeeping gene expression, setting arbitrarily a basal level (X-axis). Results are mean of triplicates±SD of two experiments, and shown on a base 2 log scale.
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f0010: Real Time PCR quantification in fresh mouse tissues: (a) and (b) GILZ and L-GILZ expression was normalized using the housekeeping gene GAPDH. All values were calculated with respect of thymus, set equal to 1 and expressed in a base 2 log scale. (c) Comparison of GILZ and L-GILZ in the same tissues as in (a) and (b) in which values were considered relatively to the housekeeping gene expression, setting arbitrarily a basal level (X-axis). Results are mean of triplicates±SD of two experiments, and shown on a base 2 log scale.

Mentions: Once designed the appropriate PCR strategy through the use of specific primers (C and D for GILZ, E and F for L-GILZ), a quantitative analysis of GILZ and L-GILZ expression in tissues were performed by Real Time PCR. Fig. 2a and b show GILZ and L-GILZ expression in a variety of fresh mouse tissues. Fig. 2c summarizes and compares the expression of both transcripts: GILZ and L-GILZ are differently expressed in almost all tissues.


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)

Real Time PCR quantification in fresh mouse tissues: (a) and (b) GILZ and L-GILZ expression was normalized using the housekeeping gene GAPDH. All values were calculated with respect of thymus, set equal to 1 and expressed in a base 2 log scale. (c) Comparison of GILZ and L-GILZ in the same tissues as in (a) and (b) in which values were considered relatively to the housekeeping gene expression, setting arbitrarily a basal level (X-axis). Results are mean of triplicates±SD of two experiments, and shown on a base 2 log scale.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0010: Real Time PCR quantification in fresh mouse tissues: (a) and (b) GILZ and L-GILZ expression was normalized using the housekeeping gene GAPDH. All values were calculated with respect of thymus, set equal to 1 and expressed in a base 2 log scale. (c) Comparison of GILZ and L-GILZ in the same tissues as in (a) and (b) in which values were considered relatively to the housekeeping gene expression, setting arbitrarily a basal level (X-axis). Results are mean of triplicates±SD of two experiments, and shown on a base 2 log scale.
Mentions: Once designed the appropriate PCR strategy through the use of specific primers (C and D for GILZ, E and F for L-GILZ), a quantitative analysis of GILZ and L-GILZ expression in tissues were performed by Real Time PCR. Fig. 2a and b show GILZ and L-GILZ expression in a variety of fresh mouse tissues. Fig. 2c summarizes and compares the expression of both transcripts: GILZ and L-GILZ are differently expressed in almost all tissues.

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.