Sample 4 ALS blood
Homo sapiens
Protocols: Peripheral blood and neuromuscular junction from a total of 70 patients with sporadic amyotrophic lateral sclerosis (n = 45), and healthy controls (n = 25) was obtained (see Table 1 Supplementary file). Blood samples from healthy controls and from patients with ALS were obtained from the Monaldi Hospital, Naples, Italy and neuromuscular junction from ALS patients were purchased from Edinburgh Tissue Bio Bank. Detailed patient characteristics are listed in Table 1 Supplementary file. ALS patients have been diagnosed as clinically definite sALS with the support of electroneuromyography (ENMG). The onset of the disease was 1-2 years before diagnosis. The mean age of patients and controls was 62 ± 10 years. RNA extraction was performed as in De Felice et al (2014). Briefly, we obtained ten micrograms of total RNA from peripheral blood samples drawn from patients and controls in the morning. Trizol (Invitrogen, no. 15596-026) method has been used for isolation and purification of RNA from whole blood and from neuromuscular junction samples. RNA was isolated including a DNase digestion step. These standardized RNA isolation procedures guarantee high-quality RNA. Using the Agilent 2100 Bioanalyzer platform (Agilent Technologies) RNA samples were quality-checked documenting the identification of 18-S and 28-S ribosomal RNA (rRNA) peaks .The yields were 8-15 μg, and the RNA Integrity Number (RIN) was between 7.2 and 10. Such results fulfilled published data, reporting that RNA with a RIN >6 is of sufficient quality for miRNA microarray experiments as well as gene and microRNA expression-profiling experiments We performed the enrichment of small RNA fraction from total RNA utilizing the Magnetic Bead Cleanup Module (Life Technologies, Carlsbad, CA, USA). Using Ion Total RNA-Seq kit v2 Life Technologies, Carlsbad, CA, USA, 50 ng of enriched small RNA sample was ligated to proper adapters and reverse transcribed to cDNA. cDNA samples were size-selected from 50 to 300 nt (Magnetic Bead Cleanup Module, Life Technologies), and PCR amplified using 5' primers containing a unique index barcode (Ion XpressTM RNA-Seq Barcode 1-16 Kit, Life Technologies, Carlsbad, CA, USA). We assessed the yield and size distribution of the small RNA libraries using the Agilent 2100 Bioanalyzer TM instrument and the Agilent HS DNA kit. We amplified similarly pooled libraries on Ion SphereTM Particles (ISPs) provided by the Life Technologies Ion PI Hi-Q Chef kit. All the steps to perform the emulsion PCR and the loading of the pooled libraries in the Life Technologies ION PI Chip v3 were performed automatically on Life Technologies ION Chef Instrument. Eight libraries were pooled in one chip.
ENA-FIRST-PUBLIC
2018-12-12T17:01:45Z
ENA-LAST-UPDATE
2018-07-25T10:25:58Z
External Id
SAMEA4803059
INSDC center name
Department of Environmental , Biological and Pharmaceutical Sciences and Technologies (DiSTABiF) University of Campania “L. Vanvitelli”
INSDC first public
2018-12-12T17:01:45Z
INSDC last update
2018-07-25T10:25:58Z
INSDC status
public
Submitter Id
E-MTAB-7073:Sample 4 ALS blood
broker name
ArrayExpress
common name
human
developmental stage
adult
disease
sporadic amyotrophic lateral sclerosis
individual
mixed pool of 45 donors
organism part
blood
sample name
E-MTAB-7073:Sample 4 ALS blood
scientific_name
Homo sapiens