Reverse transcriptase

Lumbar dorsal root ganglia (DRG; L4-L5) were dissected from adult Sprague Dawley rats, frozen in liquid nitrogen, and maintained at −80°C until processed for RNA extraction. Total RNA was extracted from the samples using the RNeasy RNA extraction and purification kit (Qiagen). Single stranded cDNA was synthesized using reverse transcriptase (Bioline) with oligo-dT primers. Quantitative PCR was performed as previously described [28] (link). Briefly, resultant cDNA samples were amplified on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) using the reporter, SYBR Green. The PCR reaction was as follows: 1x, 50°C, 2 min; 1x, 95°C, 10 min; 45x, 95°C, 15 s, 60°C, 1 min; 1x, 25°C, hold. To check for DNA contamination, PCR was run using an L27 (ribosomal housekeeping gene) primer pair, whose PCR product crosses an intron. The mRNA level for each gene (x) relative to L27 mRNA (internal control) was calculated using the following equation where Ct refers to threshold cycles: mRNA (x%) = 2^{Ct(L27)-Ct(x)}×100.
Nav 1.7 For GAGAGCGGAGAGATGGATTCNav 1.7 Rev GCTTCAGTGGTTGTGATG

RNA was isolated with Trizol reagent (Life-tecnologies). First strand cDNAs were synthesized from 1 µg of total RNA in a 20 µl reaction with reverse transcriptase according to manufacturer instructions (BioLine, London, UK). Real-time PCR was performed on 30 ng of cDNA, using a SYBR green Master Mix (Biorad, Hercules, CA, USA). β-actin was used as internal control. All primers used in these experiments are reported in Table 1.Table 1

List of primers used for RT-PCR analysis and of siRNAs for silencing experiments

Gene	Sequence
β-actin	5ʹ-AGCCATGTACGTAGCCATCC-3ʹ 5ʹ-CTCTCAGCTGTGGTGGTGAA-3ʹ
IL-1β	5ʹ-ACTCATTGTGGCTGTGGAGA-3ʹ 5ʹ-TAGCAGGTCGTCATCATCCC-3ʹ
IL-6	5ʹ-TGATGGATGCTTCCAAACTG-3ʹ 5ʹ-GAGCATTGGAAGTTGGGGTA-3ʹ
IL-8	5ʹ-GAAGATAGATTGCACCGA-3ʹ 5ʹ-CATAGCCTCTCACACATTTC-3ʹ
MCP-1	5ʹ-GCCAGTGAATGAGTAGCAAG-3ʹ 5ʹ-CTTCTGGGCCTGTTGTTCAC-3ʹ
TNF-α	5ʹACTGAACTTCGGGGTGATTG-3ʹ 5ʹGCTTGGTGGTTTGCTACGAC-5ʹ
CamKIIα siRNA#1 (27mer duplex)	rArCrArArGrArArGrArArUrGrArUrGrGrCrGrUrGrArArGrGrA
CamKIIα siRNA#2 (27mer duplex)	rGrGrCrCrUrGrGrArCrUrUrUrCrArUrCrGrArUrUrCrUrArTrT

The colonic cells were treated with Trizol (Life-technologies, Segrate, Milano, Italy) to isolate the RNA. First-strand cDNAs were synthesized from 1 µg of total RNA in a 20 µL reaction with reverse transcriptase (BioLine, Wildong Road, Memphis, TN, USA). Real-time PCR was performed using SYBR green Master Mix (Biorad, Segrate, Milano, Italy). GAPDH was used as the internal control. All primers are reported in Table 4. The relative transcription mRNA level was calculated.