#property	value
Experiment	EXR-JSAUG1UH2001-EX
- Status	Add
- exRNA Source Isolation Protocol	
-- Protocol Description	miRNA template preparation: miRNA were isolated from CSF using  mirVana PARIS, concentrated using Zymo RNA Clean & Concentrator-5, then assayed by TLDA.
-- Biofluid	
--- Cell Removal Step Done	No
- exRNA Sample Preparation Protocol	
-- Protocol Description	CSF miRNA levels were measured in AD and Control subjects using banked CSF provided by the Oregon Alzheimer's Disease Center (OADC) Biomarkers & Genetics Core. CSF samples were collected from fasting subjects in the morning using standard collection protocols consistent with other AD centers, then aliquoted and stored at -80 degC. The concentrated RNA was converted to cDNA with Megaplex RT Primers, Human Pool Set v3.0, followed by pre-amplification of each cDNA sample using Megaplex PreAmp Primers, Human Pool Set v3.0 (Life Technologies). MiRNA expression was quantified using TaqMan Low density Array (TLDA) Human MicroRNA A+B Cards Set v3.0. A QuantStudio 12K Flex Real-Time PCR System was used for RT-qPCR amplification and QuantStudio software (v1.2.2) System was used for analysis. miRNA expression was normalized within each subject to the expression of U6 small nuclear RNA (U6 snRNA).
-- Pre-purification of Extracellular Vesicles	No
-- RNA Isolation Method	
--- Description	Total RNA was isolated from 0.5 ml CSF using the mirVana PARIS kit (Life Technologies/Ambion) without enrichment for small RNAs. Isolated RNAs from each sample were concentrated using Zymo RNA Clean & Concentrator-5 (Zymo Research, Irvine CA) spin columns. RNA concentrations were measured using Quant-iT RiboGreen RNA Assay Kit (Life Technologies/Invitrogen). RNA quality was assessed using an Agilent 2100 Bioanalyzer and RNA 6000 Pico chips (Agilent Technologies).
--- RNA Isolation Kit	MiRVana Paris (Ambion)
---- Proteinase K Treatment	No
---- DNase Treatment	No
-- exRNA Quantification Method	Other
--- Other exRNA Quantification Method	Standardized CSF input volume 0.5ml
-- Enzymatic Treatment of Purified exRNA	No
-- Method for Assessment of Purity	Initial Ribogreen analylsis of a subset of samples
-- Yield	4 pg/5ml CSF
-- RNA Integrity Method	Bioanayzer on a subset of samples
--- Instrument	Agilent 2100
- Experiment Type	qPCR Assay
-- qPCR Assay	
--- Reverse Transcription	
---- Complete Reaction Conditions	As per Life Technologies Human miRNA Megaplex Pool (A and B) protocol
----- Amount of RNA	Total isolated from 0.5 ml CSF (est. TBD ng)
----- Reaction Volume	7.5 ul
----- Primary Oligonucleotide (If Using GSP)	Life Technologies Human miRNA Megaplex Pool (A and B)
----- Reverse Transcriptase	Life Technologies MultiScribe 
------ Concentration	125 U
------ Temperature	16 - 50 degC
------ Time	1 sec - 2 min
----- Manufacturer of Reagents	Life Technologies
------ Catalogue Numbers	4366596
--- Storage Conditions of cDNA	-80 C
--- qPCR Target Information	Life Technologies Human miRNA Megaplex Pool (A and B), 756 miRNA and housekeeping
---- Multiplexed?	No
---- Efficiency	Assume 100% per manufacturer
---- Sequence Accession Number	Life Technologies Human miRNA Arrays
--- qPCR Protocol	
---- Complete Reaction Conditions	Life Technologies Taqman Low Density Array Protocol
----- Reaction Volume	2.08 ul
---- Manufacturer of Plates/Tube	Life Technologies
----- Catalog Number	4444913
---- Complete Thermocycling Parameters	Per manufacturer's protocol
---- Reaction Setup	Manual
---- Manufacturer of qPCR Instrument	Life Technologies