Rneasy mini column purification kit

Total RNA was isolated from the monocytes/macrophages using the RNeasy Mini column purification kit (Qiagen) according to manufacturer’s instructions. Quantification of RNA was done by spectrophotometric analysis and OD 260/280 ratio was used to verify RNA quality. cDNA was synthesized using the Reverse Transcription system (Promega) according to the manufacturer’s instructions. The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL. Real-time quantitative PCR was performed using the CFX96 real-time detection system (Bio-Rad Laboratories). The ΔCt of the gene was normalized to the ΔCt of housekeeping gene 18S rRNA and fold change of mRNA was expressed relative to untreated cells. Human IFNβ, IP-10, TNFα, IL-6, IL-1β, IKKε, spliced XBP-1, unspliced XBP-1 primers and mouse TNFα, RANTES, HspA1A and 18S primer pairs were synthesized by IDT Inc. and enumerated in Table I. Human RANTES primer pairs were purchased from SABiosciences.

Total RNA was isolated from the monocytes/macrophages using the RNeasy Mini column purification kit (Qiagen) according to manufacturer’s instructions. RNA was quantified by spectrophotometric analysis and quality of RNA was verified by measurement of OD 260/280 ratio. cDNA was synthesized using the Reverse Transcription system (Promega) according to the manufacturer’s instructions. The reaction mixture for qPCR contained 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL. Real-time quantitative PCR was performed using the CFX96 real-time detection system (Bio-Rad Laboratories). The ΔCt of the gene was normalized to the ΔCt of housekeeping gene 18S and fold change of mRNA was expressed relative to untreated cells. Human TNFα, IL-6, IL-1β primers and mouse TNFα, IL-6, IL-1β, hsp70, HSF1 and 18S primer pairs were synthesized by IDT Inc. and enumerated in Table I. Human hsp70 and HSF1 primer pairs were purchased from SABiosciences.