|
| Status |
Public on Sep 06, 2016 |
| Title |
Comparison of healthy donors versus Rheumatoid arthritis patients |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Blood of healthy donors
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: fresh peripheral blood
disease status: healthy donor with no history of autoimmune disease
|
| Biomaterial provider |
Rheumatology department at the university hospital of Montpellier
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Blood samples were collected using EDTA-coated tubes (BD VacutainerTM 5 ml; BD Diagnostics, France) according to standard procedure. Aliquots of 0.5 ml of blood samples were immediately transferred to 1.2 ml of RNAlater medium (Applied Biosystems) and stored at -20°C. Total RNA was extracted using a modified protocol from the Ribopure-Blood RNA isolation kit (Applied Biosystems). Briefly, 10µl glacial acid (Sigma, France) was added to blood cell lysate (800µl, step 1 and 2 according to the manufacturer’s instruction). The samples were extracted with acid phenol/Chloroform, 1 ml of GuSCN Lysis solution (4 M Guanidinium Thiocyanate, 25 mM Sodium Citrate, 0.5%(w/v) Sodium N-lauroyl Sarcosinate and 0.1 M beta-mercaptoethanol and 1.25 volumes of ethanol were added to the aqueous phase. The samples were passed through a Filter cartridge and washed first with wash solution 1 (70% EtOH/30%GuSCN lysis solution) and second with wash solution 2 (80% EtOH/ 50 mM NaCl). The RNA was eluted in 100µl Elution solution preheated to 80°C and stored at -20°C. The concentration and integrity of RNA were determined by NanoDrop ND-1000 spectrophotometry (NanoDrop Tech, Rockland, Del) and by a Bioanalyzer Agilent 1. For pool samples, measures were taken to guarantee that RNA from each subject was of equal amount.
|
| Label |
Cy3
|
| Label protocol |
The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments.
|
| |
|
| Channel 2 |
| Source name |
Blood of Rheumatoid arthritis patients
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: fresh peripheral blood
disease status: Rheumatoid arthritis patients fulfilling the 1987 revised classification criteria of the american college of rheumatology.
therapy: oral corticosteroids for at least one month
|
| Biomaterial provider |
Rheumatology department at the university hospital of Montpellier
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Blood samples were collected using EDTA-coated tubes (BD VacutainerTM 5 ml; BD Diagnostics, France) according to standard procedure. Aliquots of 0.5 ml of blood samples were immediately transferred to 1.2 ml of RNAlater medium (Applied Biosystems) and stored at -20°C. Total RNA was extracted using a modified protocol from the Ribopure-Blood RNA isolation kit (Applied Biosystems). Briefly, 10µl glacial acid (Sigma, France) was added to blood cell lysate (800µl, step 1 and 2 according to the manufacturer’s instruction). The samples were extracted with acid phenol/Chloroform, 1 ml of GuSCN Lysis solution (4 M Guanidinium Thiocyanate, 25 mM Sodium Citrate, 0.5%(w/v) Sodium N-lauroyl Sarcosinate and 0.1 M beta-mercaptoethanol and 1.25 volumes of ethanol were added to the aqueous phase. The samples were passed through a Filter cartridge and washed first with wash solution 1 (70% EtOH/30%GuSCN lysis solution) and second with wash solution 2 (80% EtOH/ 50 mM NaCl). The RNA was eluted in 100µl Elution solution preheated to 80°C and stored at -20°C. The concentration and integrity of RNA were determined by NanoDrop ND-1000 spectrophotometry (NanoDrop Tech, Rockland, Del) and by a Bioanalyzer Agilent 1. For pool samples, measures were taken to guarantee that RNA from each subject was of equal amount.
|
| Label |
Cy5
|
| Label protocol |
The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 mL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C. After RNA hybridization, tag-conjugating Cy3 and Cy5 dyes were circulated through the microfluidic chip for dye staining.
|
| Scan protocol |
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
Additional eligibility criteria: methotrexate (MTX) treatment; DAS28 ≥ 3.2; and resistance to at least 2 DMARDs (MTX and anti-TNF included). For one month or more before the start of this study, every patient was given stable doses of oral corticosteroids and did not receive any intra-articular steroid injections.
|
| Data processing |
Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression).
|
| |
|
| Submission date |
Sep 14, 2009 |
| Last update date |
Sep 06, 2016 |
| Contact name |
Florence Apparailly |
| E-mail(s) |
florence.apparailly@inserm.fr
|
| Phone |
33 499 636 086
|
| Fax |
33-499-636-020
|
| Organization name |
INSERM
|
| Department |
u844
|
| Street address |
CHU Saint Eloi, 80 rue Augustin Fliche
|
| City |
Montpellier |
| ZIP/Postal code |
34295 |
| Country |
France |
| |
|
| Platform ID |
GPL8366 |
| Series (1) |
| GSE18097 |
Comparison of miRNA from healthy donors versus Rheumatoid arthritis patients |
|
