Comparison of blood RNA extraction methods used for gene expression profiling in amyotrophic lateral sclerosis.

Author(s): Bayatti N, Cooper-Knock J, Bury JJ, Wyles M, Heath PR, Kirby J, Shaw PJ

Publication: PLoS One, 2014, Vol. 9, Page e87508

PubMed ID: 24475299 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare gene expression profiling between specimens in the PAX, TEM, and LL blood collection and RNA extraction systems, and to determine the utility of globin depletion with GlobinClear. Specimens were obtained from 8 sporadic amyotrophic lateral sclerosis (ALS) patients and 7 healthy volunteers. PAX and TEM systems use their own collection tubes, but specimens intended for LL processing were collected into K2EDTA tubes, passed through LL filter, and RNAlater was added. All specimens were frozen at -20 degrees C until analysis.

   Summary of Findings:

   The highest quantity of RNA was extracted using the TEM system, followed by the PAX system, and lastly, the LL system. TEM and LL-extracted RNA showed high quality (RIN >7.0) without DNase treatment. However, PAX-extracted RNA needed to undergo DNase treatment before high quality RNA was obtained. This DNase treatment also decreased the yield of RNA from PAX-extracted specimens. Further, globin mRNA depletion by GC lowered the RIN values for PAX-extracted RNA (PAX RIN=7.9, PAX GC RIN=7.2) but not for TEM-extracted RNA (both RIN=7.9). In general, PAX RNA array hybridizations showed the most variability. GC treatment increased the microarray percent present calls for PAX and TEM-extracted RNA from 39% and 43%, respectively, to 50% for each, the same as LL-extracted RNA. In addition, globin clearance unmasked transcripts that were otherwise not detected (6022 probe sets). The PAX GC RNA and TEM GC RNA had more differentially-regulated probe sets between ALS patients and healthy controls in common than either did with LL RNA. For both TEM GC specimens and LL specimens, 4 out of 6 genes chosen for validation of differential expression between ALS patients and healthy controls were indeed validated. The authors report that PAX GC specimens were not used for validation due to the longer preparation time required.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • Other Preservative
   • PAXgene

   Diagnoses:

   • Normal
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	PAXgene collection tube TEMPUS collection tube K2EDTA Vacutainer
   Preaquisition	Diagnosis/ patient condition	Amyotrophic lateral sclerosis Healthy
   Analyte Extraction and Purification	Analyte isolation method	PAX TEM LL
   Analyte Extraction and Purification	Analyte purification	Globin depletion No globin depletion
   Analyte Extraction and Purification	Nucleic acid digestion	DNase treatment No DNase treatment

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