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Bypassing hazard of housekeeping genes: their evaluation in rat granule neurons treated with cerebrospinal fluid of multiple sclerosis subjects.

Mathur D, Urena-Peralta JR, Lopez-Rodas G, Casanova B, Coret-Ferrer F, Burgal-Marti M - Front Cell Neurosci (2015)

Bottom Line: Appropriate normalization of target gene transcript(s) based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results.NormFinder identified Tfrc as the best invariable gene followed by B2m and Rpl19.ActB and Gapdh are not recommended in gene expression studies related to current one.

View Article: PubMed Central - PubMed

Affiliation: Department of Functional Biology, University of Valencia Valencia, Spain ; Multiple Sclerosis Laboratory, Department of Biomedicine, Prince Felipe Research Center Valencia, Spain.

ABSTRACT
Gene expression studies employing real-time PCR has become an intrinsic part of biomedical research. Appropriate normalization of target gene transcript(s) based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results. In multiple sclerosis (MS), several gene expression studies have been undertaken, however, the suitability of housekeeping genes to express stably in this disease is not yet explored. Recent research suggests that their expression level may vary under different experimental conditions. Hence it is indispensible to evaluate their expression stability to accurately normalize target gene transcripts. The present study aims to evaluate the expression stability of seven housekeeping genes in rat granule neurons treated with cerebrospinal fluid of MS patients. The selected reference genes were quantified by real time PCR and their expression stability was assessed using GeNorm and NormFinder algorithms. GeNorm identified transferrin receptor (Tfrc) and microglobulin beta-2 (B2m) the most stable genes followed by ribosomal protein L19 (Rpl19) whereas β-actin (ActB) and glyceraldehyde-3-phosphate-dehydrogenase (Gapdh) the most fluctuated ones in these neurons. NormFinder identified Tfrc as the best invariable gene followed by B2m and Rpl19. ActB and Gapdh were the least stable genes as analyzed by NormFinder algorithm. Both methods reported Tfrc and B2m the most stably expressed genes and Gapdh the least stable one. Altogether our data demonstrate the significance of pre-validation of housekeeping genes for accurate normalization and indicates Tfrc and B2m as best endogenous controls in MS. ActB and Gapdh are not recommended in gene expression studies related to current one.

No MeSH data available.


Related in: MedlinePlus

(A) Immunofluorescence of primary cultures of CGNs. (a) DAPI stained nuclei. Neurofilaments stained with (b) Texas red and (c) FITC dyes. (d) Merged images. (B) Analysis of β-actin (a) and Gapdh (b) expression by PCR: (1) Electrophoretic bands; (2) Integrated density obtained by Image J software quantification. The values correspond with the fold change vs. control values of the MS clinical forms, NMO, and control patients.
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Figure 2: (A) Immunofluorescence of primary cultures of CGNs. (a) DAPI stained nuclei. Neurofilaments stained with (b) Texas red and (c) FITC dyes. (d) Merged images. (B) Analysis of β-actin (a) and Gapdh (b) expression by PCR: (1) Electrophoretic bands; (2) Integrated density obtained by Image J software quantification. The values correspond with the fold change vs. control values of the MS clinical forms, NMO, and control patients.

Mentions: Cerebellar granule neurons were stained with Texas Red and FITC dyes. The nuclei of neurofilaments were stained with DAPI. Figure 2A shows pure cultures of granule neurons isolated from cerebellum with stained neurofilaments.


Bypassing hazard of housekeeping genes: their evaluation in rat granule neurons treated with cerebrospinal fluid of multiple sclerosis subjects.

Mathur D, Urena-Peralta JR, Lopez-Rodas G, Casanova B, Coret-Ferrer F, Burgal-Marti M - Front Cell Neurosci (2015)

(A) Immunofluorescence of primary cultures of CGNs. (a) DAPI stained nuclei. Neurofilaments stained with (b) Texas red and (c) FITC dyes. (d) Merged images. (B) Analysis of β-actin (a) and Gapdh (b) expression by PCR: (1) Electrophoretic bands; (2) Integrated density obtained by Image J software quantification. The values correspond with the fold change vs. control values of the MS clinical forms, NMO, and control patients.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585208&req=5

Figure 2: (A) Immunofluorescence of primary cultures of CGNs. (a) DAPI stained nuclei. Neurofilaments stained with (b) Texas red and (c) FITC dyes. (d) Merged images. (B) Analysis of β-actin (a) and Gapdh (b) expression by PCR: (1) Electrophoretic bands; (2) Integrated density obtained by Image J software quantification. The values correspond with the fold change vs. control values of the MS clinical forms, NMO, and control patients.
Mentions: Cerebellar granule neurons were stained with Texas Red and FITC dyes. The nuclei of neurofilaments were stained with DAPI. Figure 2A shows pure cultures of granule neurons isolated from cerebellum with stained neurofilaments.

Bottom Line: Appropriate normalization of target gene transcript(s) based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results.NormFinder identified Tfrc as the best invariable gene followed by B2m and Rpl19.ActB and Gapdh are not recommended in gene expression studies related to current one.

View Article: PubMed Central - PubMed

Affiliation: Department of Functional Biology, University of Valencia Valencia, Spain ; Multiple Sclerosis Laboratory, Department of Biomedicine, Prince Felipe Research Center Valencia, Spain.

ABSTRACT
Gene expression studies employing real-time PCR has become an intrinsic part of biomedical research. Appropriate normalization of target gene transcript(s) based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results. In multiple sclerosis (MS), several gene expression studies have been undertaken, however, the suitability of housekeeping genes to express stably in this disease is not yet explored. Recent research suggests that their expression level may vary under different experimental conditions. Hence it is indispensible to evaluate their expression stability to accurately normalize target gene transcripts. The present study aims to evaluate the expression stability of seven housekeeping genes in rat granule neurons treated with cerebrospinal fluid of MS patients. The selected reference genes were quantified by real time PCR and their expression stability was assessed using GeNorm and NormFinder algorithms. GeNorm identified transferrin receptor (Tfrc) and microglobulin beta-2 (B2m) the most stable genes followed by ribosomal protein L19 (Rpl19) whereas β-actin (ActB) and glyceraldehyde-3-phosphate-dehydrogenase (Gapdh) the most fluctuated ones in these neurons. NormFinder identified Tfrc as the best invariable gene followed by B2m and Rpl19. ActB and Gapdh were the least stable genes as analyzed by NormFinder algorithm. Both methods reported Tfrc and B2m the most stably expressed genes and Gapdh the least stable one. Altogether our data demonstrate the significance of pre-validation of housekeeping genes for accurate normalization and indicates Tfrc and B2m as best endogenous controls in MS. ActB and Gapdh are not recommended in gene expression studies related to current one.

No MeSH data available.


Related in: MedlinePlus