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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) Immunodetection of oligoclonal bands (OCBs) in serum (S) and cerebrospinal fluid (CSF) (L). Pattern 1: No OCBs seen (negative, polyclonal): No OCBs in CSF or serum. No intrathecal Ig synthesis; Pattern 2: OCBs in CSF only (positive): OCBs present in CSF only. Intrathecal IgG synthesis as seen in MS; Pattern 3: Identical OCBs in both (mirror images): Bands in serum mirror those in CSF. This suggests systemic Ig synthesis; Pattern 4: Identical OCBs in both with extra bands in CSF: Identical bands in both serum and CSF with extra bands in CSF. Image demonstrates both intrathecal and systemic Ig synthesis. This is identical as it is seen in MS. (B) Indirect immunofluorescence in cells transfected by aquaporin 4 (EUROIMMUN Aquaporin-4 IIFT). Panel I: Anti-AQP4 antibodies observed in the serum of NMO patients (positive sample); Panel II: Absence of anti-AQP4 antibodies in serum sample (negative sample).
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Figure 1: (A) Immunodetection of oligoclonal bands (OCBs) in serum (S) and cerebrospinal fluid (CSF) (L). Pattern 1: No OCBs seen (negative, polyclonal): No OCBs in CSF or serum. No intrathecal Ig synthesis; Pattern 2: OCBs in CSF only (positive): OCBs present in CSF only. Intrathecal IgG synthesis as seen in MS; Pattern 3: Identical OCBs in both (mirror images): Bands in serum mirror those in CSF. This suggests systemic Ig synthesis; Pattern 4: Identical OCBs in both with extra bands in CSF: Identical bands in both serum and CSF with extra bands in CSF. Image demonstrates both intrathecal and systemic Ig synthesis. This is identical as it is seen in MS. (B) Indirect immunofluorescence in cells transfected by aquaporin 4 (EUROIMMUN Aquaporin-4 IIFT). Panel I: Anti-AQP4 antibodies observed in the serum of NMO patients (positive sample); Panel II: Absence of anti-AQP4 antibodies in serum sample (negative sample).

Mentions: Anti-AQP4 antibody in NMO has a high specificity so as to contribute to early diagnosis and optimized treatment of Devic disease. Serum sample diluted 1:10 in PBS-Tween was used to detect the presence of NMO specific IgG antibodies. Indirect immunofluorescence (IFI) was performed to diagnose NMO (Figure 1B). Antibodies against aquaporin 4 were detected using a cell line, which was molecular biologically modified to produce large quantities of aquaporin 4. In this method (EuroImmun IIFT) recombinantly transfected cells act as an antigen substrate to be incubated with diluted serum samples for half an hour.


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) Immunodetection of oligoclonal bands (OCBs) in serum (S) and cerebrospinal fluid (CSF) (L). Pattern 1: No OCBs seen (negative, polyclonal): No OCBs in CSF or serum. No intrathecal Ig synthesis; Pattern 2: OCBs in CSF only (positive): OCBs present in CSF only. Intrathecal IgG synthesis as seen in MS; Pattern 3: Identical OCBs in both (mirror images): Bands in serum mirror those in CSF. This suggests systemic Ig synthesis; Pattern 4: Identical OCBs in both with extra bands in CSF: Identical bands in both serum and CSF with extra bands in CSF. Image demonstrates both intrathecal and systemic Ig synthesis. This is identical as it is seen in MS. (B) Indirect immunofluorescence in cells transfected by aquaporin 4 (EUROIMMUN Aquaporin-4 IIFT). Panel I: Anti-AQP4 antibodies observed in the serum of NMO patients (positive sample); Panel II: Absence of anti-AQP4 antibodies in serum sample (negative sample).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: (A) Immunodetection of oligoclonal bands (OCBs) in serum (S) and cerebrospinal fluid (CSF) (L). Pattern 1: No OCBs seen (negative, polyclonal): No OCBs in CSF or serum. No intrathecal Ig synthesis; Pattern 2: OCBs in CSF only (positive): OCBs present in CSF only. Intrathecal IgG synthesis as seen in MS; Pattern 3: Identical OCBs in both (mirror images): Bands in serum mirror those in CSF. This suggests systemic Ig synthesis; Pattern 4: Identical OCBs in both with extra bands in CSF: Identical bands in both serum and CSF with extra bands in CSF. Image demonstrates both intrathecal and systemic Ig synthesis. This is identical as it is seen in MS. (B) Indirect immunofluorescence in cells transfected by aquaporin 4 (EUROIMMUN Aquaporin-4 IIFT). Panel I: Anti-AQP4 antibodies observed in the serum of NMO patients (positive sample); Panel II: Absence of anti-AQP4 antibodies in serum sample (negative sample).
Mentions: Anti-AQP4 antibody in NMO has a high specificity so as to contribute to early diagnosis and optimized treatment of Devic disease. Serum sample diluted 1:10 in PBS-Tween was used to detect the presence of NMO specific IgG antibodies. Indirect immunofluorescence (IFI) was performed to diagnose NMO (Figure 1B). Antibodies against aquaporin 4 were detected using a cell line, which was molecular biologically modified to produce large quantities of aquaporin 4. In this method (EuroImmun IIFT) recombinantly transfected cells act as an antigen substrate to be incubated with diluted serum samples for half an hour.

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