Ge 96.96 dynamic arrays

Differential expression levels of 48 identified DEGs (SDC-2) in serum SEVs of people living with DLB were evaluated using high-throughput quantitative polymerase chain reactions (qPCR). Total SEV RNA were purified using the Invitrogen total SEV RNA isolation kit (Thermo Fisher Scientific, USA) from 0.5ml aliquots of serum samples that had been sequenced (N=20). 100ng of SEV RNA/sample were reverse transcribed using the miScript ® -II RT Kit and its 5X miScript HiFlex buffer (Qiagen, UK). After 19 cycles of specific target amplification with the PreAmp master-mix (Fluidigm, USA), high-throughput qPCR was performed using the BioMark HD, GE 96.96 dynamic arrays (Fluidigm, USA), and SsoFast EvaGreen low ROX kit (Bio-Rad, USA). SDC-3 provides further details of the qPCR verification. A DEG would be verified if it met the following criteria ( 34): (i) Both RNA-Seq and qPCR showed same direction of differential expression, and (ii) differential expression fold change, estimated by qPCR, was either above 1.25 or below 0.80 (logarithmic fold change (L2FC) cut-off ±0.3219).

Differential expression of 78 selected genes (SDC-4) including all protein coding FDRadjusted DEGs and 10 randomly selected DEGs (edgeR p<0.05; no df) in DLPFC of LBD brains were evaluated using high-throughput quantitative polymerase chain reactions (qPCR).
One µg of RNA per sample from the aliquots of RNA that had been sequenced (N=40) were reverse transcribed using the iScript™ advanced cDNA synthesis kit (Bio-Rad, Hercules, USA). After 14 cycles of specific target amplification with the PreAmp master-mix (Fluidigm, San Francisco, USA), high-throughput qPCR was performed using the BioMark HD, GE 96.96 dynamic arrays (Fluidigm, San Francisco, USA), and SsoFast EvaGreen low ROX kit (Bio-Rad, Hercules, USA) (SDC-5). Verification of differential expression of a gene was defined by the following criteria ( 26), (i) Both RNA-seq and qPCR showed same direction of differential expression, (ii) Differential expression fold change, estimated by qPCR, was either above 1.25 or below 0.80 (logarithmic cut-off was ±0.3219).