Rna total cleanup kits

The total RNA isolated from given samples was purified using the QIAGEN RNA total cleanup kits as per manufacturers instruction. One hundred nanogram of the RNA was converted using EvaGreen RT conversion kit in a gradient thermocycler as per manufacturers description (ABMbio, USA). One nanogram of the converted cDNA samples was added to Bright-Green SYBR qPCR master-mix and qPCR analysis was performed in an ABI StepOne Plus PCR system. The PCR conditions were as follows: 95 °C for 1 min followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and with a final round of extension at 72 °C for 10 min^{15 (link)}. Sequences of primers used are detailed in Table 1.Table 1

qPCR genes of interest, sense and antisense primer sequence and amplicon size.

Gene	Forward primer 5′-3’	Reverse primer 5′-3’	Size	Refs.
CXCL10	GAAAGCAGTTAGCAAGGAAAGGTC	ATGTAGGGAAGTGATGGGAGAGG	120	16 (link)
CCR7	TGGTGGTGGCTCTCCTTGTC	TGTGGTGTTGTCTCCGATGTAATC	83	16 (link)
IL-12	AAAGGACATCTGCGAGGAAAGTTC	CGAGGTGAGGTGCGTTTATGC	128	16 (link)
CD206	ACCTCACAAGTATCCACACCATC	CTTTCATCACCACACAATCCTC	213	16 (link)
CD163	GTCGCTCATCCCGTCAGTCATC	GCCGCTGTCTCTGTCTTCGC	114	16 (link)
CCL17	GAGCCATTCCCCTTAGAAAG	AGGCTTCAAGACCTCTCAAG	172	16 (link)
NFκB (RELA)	CCAGACCAACAACAACCCCT	TCACTCGGCAGATCTTGAGC	197	17 (link),18 (link)
IRF3	TCTGCCCTCAACCGCAAAGAAG	TACTGCCTCCACCATTGGTGTC	151	17 (link)

The total RNA isolated from given samples was purified using the QIAGEN RNA total cleanup kits as per manufacturers instruction. One hundred nanogram of the RNA was converted using EvaGreen RT conversion kit in a gradient thermocycler as per manufacturers description. One nanogram per microliter of the converted cDNA samples was added to EvaGreen SYBR qPCR master-mix and qPCR analysis was performed in an ABI StepOne Plus PCR system. Annealing temperatures were kept at 60°C, and 40 cycles of amplification were performed to produce a sufficient read. Sequences of primers used are detailed in Table 2.