Ignite Creation Date:
2024-05-06 @ 8:11 AM
Last Modification Date:
2024-10-26 @ 11:57 AM
Study NCT ID:
NCT02689115
Status:
COMPLETED
Last Update Posted:
2016-02-23
First Post:
2016-02-15
Brief Title:
NFKB1 and IKK Epsilon in Rheumatoid Arthritis
Sponsor:
Asociación Científica Latina AC
Organization:
Asociación Científica Latina AC
Study Overview
Official Title:
NF-KB1IKK Epsilon Genetic Expression in Patients With Rheumatoid Arthritis
Status:
COMPLETED
Status Verified Date:
2016-02
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
NUIRA
Brief Summary:
Rheumatoid arthritis RA is a systemic and auto-immune disorder whose primary characteristic is the chronic inflammation of joints The objective of this study was to evaluate whether there was an association between the NF-KB1IKK epsilon genetic expression and the clinical activity in RA
60 RA patients were included in the study 30 with clinical activity and 30 with clinical remission The NF-KB1IKK epsilon genetic expression was performed by real time quantitative Polymerase chain reaction qPCR through the Pfaffl method of relative quantification with Taqman probes
60 RA patients were included in the study 30 with clinical activity and 30 with clinical remission The NF-KB1IKK epsilon genetic expression was performed by real time quantitative Polymerase chain reaction qPCR through the Pfaffl method of relative quantification with Taqman probes
Detailed Description:
Patients with a confirmed diagnosis of RA were allocated in two groups based on the Disease Activity Score 28 DAS28 a with clinical activity and b with clinical remission Key exclusion criteria were patients with any other inflammatory or autoimmune disease and patients with infections The control group was represented by healthy patients Sample size
The sample size calculation was carried out through the following formula
n Nz2 pq d2N-1 z2 pq With N30 Z246 99 of confidence p09 q01 d005 95 of accuracy The sample size contained 20 patients with RA
Anthropometric measurements Weight kg and height m were calculated in a mechanical column scale SECA The patients were classified according to their Body mass index BMI weight Kg height m2 as a normal weight BMI 249 b overweight 249 kgm2 BMI299 kgm2 and c obese BMI 30 kgm2
Lymphocyte extraction Lymphocytes from peripheral blood were extracted using the ACK Lysing Buffer Life technologies Grand Island NY kit Briefly a venous blood sample in EDTA tube BD Vacutainer Franklin Lakes NJ was centrifuged at 3500 rpm during 5-8 minutes The resulting intermediate white colored phase was extracted and placed in an eppendorf tube 1 mL of ACK Lysing Buffer was added and carefully re-suspended Once again the suspension was centrifuged at 3500 rpm during 5-8 minutes and the supernatant was discarded This last step was repeated until the leukocyte package was completely white Finally it was added 100 mcl of ACK Lysing Buffer and frozen at -70ºC for later use
Genetic expression From the leukocyte package approximately 10 to 15 mg it was performed a messenger RNA mRNA extraction using the Magna Pure LC RNA isolation kit III Roche in the Magna Pure LC 20 Instrument The A260A280 nm absorbance ratio was 18 quality and total RNA concentration was calculated by determining absorbance at 260 nm established with the NanoPhotometer Implen GmbH Germany and the extracts were adjusted to a concentration of 20 µg of DNA for the PCR reaction
Subsequently the cDNA was synthesized with the Transcriptor High Fidelity cDNA Synthesis Kit Roche Applied Science It was performed a real time polymerase chain reaction qPCR using the specific primers and probes for NF-KB1 IKK epsilon and 18s as constitutive gene using a 7500 Fast Real Time PCR System Applied Biosystems Applera UK Cheshire UK mixing TaqMan Universal PCR Master Mix and the specific probes for each gene NF-KB1 Hs00765730-m1 IKK epsilon Hs01063858-m1 and 18S Hs99999901-s1 Applied Biosystems Applera UK Cheshire UK
According to the Ct obtained from the two different groups it was calculated the relative units of expression through the Pfaffl relative quantification method in which it was used as control a healthy patient
Statistical analysis The statistical analysis was performed through the Sigmaplot software version 120 Parametric and non-parametric test were used in function to the variable distribution among the parametric test that were performed to compare the groups the Students T-test was included the non-parametric tests applied were The Mann-Whitney U test and the Shapiro-Wilk test Finally a receiver operating characteristic ROC was used for the analysis of the genetic expression of NF-KB1IKK epsilon with the SPSS software version 220
The sample size calculation was carried out through the following formula
n Nz2 pq d2N-1 z2 pq With N30 Z246 99 of confidence p09 q01 d005 95 of accuracy The sample size contained 20 patients with RA
Anthropometric measurements Weight kg and height m were calculated in a mechanical column scale SECA The patients were classified according to their Body mass index BMI weight Kg height m2 as a normal weight BMI 249 b overweight 249 kgm2 BMI299 kgm2 and c obese BMI 30 kgm2
Lymphocyte extraction Lymphocytes from peripheral blood were extracted using the ACK Lysing Buffer Life technologies Grand Island NY kit Briefly a venous blood sample in EDTA tube BD Vacutainer Franklin Lakes NJ was centrifuged at 3500 rpm during 5-8 minutes The resulting intermediate white colored phase was extracted and placed in an eppendorf tube 1 mL of ACK Lysing Buffer was added and carefully re-suspended Once again the suspension was centrifuged at 3500 rpm during 5-8 minutes and the supernatant was discarded This last step was repeated until the leukocyte package was completely white Finally it was added 100 mcl of ACK Lysing Buffer and frozen at -70ºC for later use
Genetic expression From the leukocyte package approximately 10 to 15 mg it was performed a messenger RNA mRNA extraction using the Magna Pure LC RNA isolation kit III Roche in the Magna Pure LC 20 Instrument The A260A280 nm absorbance ratio was 18 quality and total RNA concentration was calculated by determining absorbance at 260 nm established with the NanoPhotometer Implen GmbH Germany and the extracts were adjusted to a concentration of 20 µg of DNA for the PCR reaction
Subsequently the cDNA was synthesized with the Transcriptor High Fidelity cDNA Synthesis Kit Roche Applied Science It was performed a real time polymerase chain reaction qPCR using the specific primers and probes for NF-KB1 IKK epsilon and 18s as constitutive gene using a 7500 Fast Real Time PCR System Applied Biosystems Applera UK Cheshire UK mixing TaqMan Universal PCR Master Mix and the specific probes for each gene NF-KB1 Hs00765730-m1 IKK epsilon Hs01063858-m1 and 18S Hs99999901-s1 Applied Biosystems Applera UK Cheshire UK
According to the Ct obtained from the two different groups it was calculated the relative units of expression through the Pfaffl relative quantification method in which it was used as control a healthy patient
Statistical analysis The statistical analysis was performed through the Sigmaplot software version 120 Parametric and non-parametric test were used in function to the variable distribution among the parametric test that were performed to compare the groups the Students T-test was included the non-parametric tests applied were The Mann-Whitney U test and the Shapiro-Wilk test Finally a receiver operating characteristic ROC was used for the analysis of the genetic expression of NF-KB1IKK epsilon with the SPSS software version 220
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None