Ribopure blood rna isolation kit

For both small pilot studies in Doha, Qatar and the Western and South Central USA, total cellular RNA was isolated using the Ribopure™ - Blood RNA Isolation Kit (Thermo Fisher) according to manufacturer's protocol and eluted in two 50 µl aliquots. Prior to removing the stabilized blood from the collection tube, the 50 ml conical tube holding the homeRNA device was briefly centrifuged 10 s at 50 g. Isolated RNA was stored at −80°C until ready for further analysis.

The blood sera were obtained as mentioned above. The levels of TNF-α, IL-1β, and IL-6 in the sera of CFA-induced animals were determined using an ELISA kit (Sigma Bioscience, USA), according to the manufacturer’s instructions.
Total blood RNA was extracted using the RiboPure™ Blood RNA Isolation kit (Thermo Fisher Scientific, USA). Geneious software (USA) was used for designing primers for qRT-PCR. The following primers were used for qRT-PCR: IL-6 (5′-CATTCTGTCTCGAGCCCACC-3′, 5′-GCAACTGGCTGGAAGTCTCT-3′); TNF-α, (5′-CTGAAGTCTGCGTCTGTCGT-3′, 5′-GTTCCACAGGGGTCTTGGAG-3′); IL-1β (5′-CCTCTGCCTCTTGACGATGG-3′, 5′-AGGACGTGCGGCAAGTATAG-3′). GAPDH (5′-GTGCCAGCCTCGTCTCATAG-3′, 5′-AGAGAAGGCAGCCCTGGTAA-3′) was used as an internal control. Three technical replicates for each biological replicate were used. RNA was quantified using Qubit fluorometer (Thermo Fisher).
The following components were added tothe PCR master-mix: 1.5 μL cDNA, 1 μL (5 pm/μL) each primer, and 5 μL DyNAmo Flash SYBR Green (Thermo Fisher) (2×). The PCR was cycled 42 times with the following conditions: 10 s at 95°C, 40 cycles for 22 s at 95°C, 60 s at 60°C. ABI Prism 7500 (Applied Biosystems, USA) was used for the qRT-PCR run. The Ct (threshold cycle) value was normalized and quantified using the Ct value of GADPH. The 2^−ΔΔCt method (27 (link)) was used to calculate the relative expression.