Ez1 rna cell mini kit

Total RNA was extracted from the cells using an EZ1 RNA Cell Mini Kit (QIAGEN, Valencia,
CA, USA). Total RNA was reverse transcribed with a random primer using Superscript reverse
transcriptase (Life Technologies), according to the manufacturer’s instructions. One-tenth
of the cDNA was amplified with Th17-related factors specific primers by PCR with 2.5 U of
Taq DNA polymerase (Takara Bio., Kusatsu, Japan) in a total volume of
50 µl, according to the manufacturer’s instructions. The PCR cycles were
allowed to run for 30 s at 94°C, followed by 30 s at 60°C and 30 s at 72°C. Before the
first cycle, a denaturation step of 1 min at 94°C was included; and after 40 cycles, the
extension was prolonged for 3 min at 72°C. Then 10-µl aliquots of each
PCR product were electrophoresed through 3% NuSieve 3:1 agarose gel (Takara Bio) and
visualized by ethidium bromide staining. The primers used were as follows: mouse
Il17a, forward, 5’-CTC CAG AAG GCC CTC AGA CTA C-3’, and reverse,
5’-TGG AGG GCA GAC AAT TCT GAA TC-3’; Il17f, forward, 5’-GAG GAT AAC ACT
GTG AGA GTT GAC-3’, and reverse, 5’-GCT TGG TGG ACA ATG GGC TTG-3’;
Il23r, 5’-TCA GTG CTA CAA TCT TCA GAG GAC A-3’, and reverse, 5’-CAA CAT
TCC TAG AGG ACA GTC TC-3’; Rorc, 5’-CCG CTG AGA GGG CTT CAC-3’, and
reverse, 5’-CTT GAC AGC ATC TCG GGA CAT G-3’; and Gapgh, 5’-TGC TGA GTA
TGT CGT GGA GTC TA-3’, and reverse, 5’-AGT GGG AGT TGC TGT TGA AGT CG-3’.

Total RNA was extracted from the cells using an EZ1 RNA Cell Mini Kit (QIAGEN). Total RNA
was reverse transcribed with a random primer using Superscript reverse transcriptase (Life
Technologies), according to the manufacturer’s instructions. The reverse transcriptase
products were then subjected to quantitative PCR (qPCR) in a StepOne realtime PCR system
(Applied Biosystems, Foster City, CA, USA) using Universal ProbeLibrary Probes (Roche,
Basel, Switzerland) and specific primers for each gene. The primers used were as follows:
mouse Rag1, forward, 5’-AGG CCT GTG GAG CAA GGT A-3’, and reverse, 5’-GCT
CAG GGT AGA CGG CAA G-3’; Rag2, forward, 5’-TGC CAA AAT AAG AAA GAG TAT
TTC AC-3’, and reverse, 5’-GGG ACA TTT TTG ATT GTG AAT AGG-3’; terminal deoxynucleotidyl
transferase (Tdt), 5’-TGG GGA GAC ATC AGC TTG TT-3’, and reverse, 5’-GGC
AAG CGT ACT GGG AGA T-3’; and Gapgh, 5’-GAG CCA AAC GGG TCA TCA-3’, and
reverse, 5’-CAT ATT TCT CGT GGT TCA CAC C-3’. The Universal ProbeLibrary Probes used were
as follows: mouse Rag1, #46; Rag2, #4;
Tdt, #62; and Gapgh, #29.

Total RNA was isolated as described previously [66] (link), [37] (link). After treatment cells were immediately lysed in Buffer RLT (Qiagen; Valencia, CA) supplemented with β-mercaptoethanol, homogenized by QIAshredder spin column (Qiagen), and kept in a −80 °C freezer until further processing. Total RNA was isolated on the EZ1 Advanced XL (Qiagen) automated RNA purification instrument using the EZ1 RNA Cell Mini Kit (Qiagen) following the manufacturer’s protocol, including an on-column DNase digestion. RNA concentration and purity (260/280 ratio) were measured with the NanoDrop 2000 UV–vis spectrophotometer (NanoDrop Products, Wilmington, DE). Integrity of RNA samples was assessed by the Agilent 2100 Bioanalyzer (Santa Clara, CA) with the RNA 6000 Nano Reagent Kit from the same manufacturer. The threshold of acceptability for Bioanalyzer RIN values was ≥8.5 for the RNA samples.

The 20 nm and 50 nm BioPure^® silver nanoparticle citrate solution was purchased from nanoComposix (San Diego, CA). The human colon carcinoma Caco2 cells (ATCC HTB-37), were obtained from the American Type Culture Collection (ATCC), Manassas, VA. Deep-frozen vials of stock cells were routinely stored in liquid nitrogen freezer. Dulbecco’s modified Eagle’s medium (DMEM) GlutaMax, Hanks’ balanced salt solution (HBSS), HEPES, phosphate buffered saline (PBS), trypsin-EDTA solution and 0.4% trypan blue solution were purchased from Invitrogen Corporation (Grand Island, NY). Fetal bovine serum (FBS) was purchased from the Hyclone Labs (Logan, UT). The sterile nonpyrogenic polystyrene cell culture flasks and plates were purchased from Corning (Corning, NY) and Becton-Dickinson (Franklin Lakes, NJ), respectively. All other chemicals were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO). Buffer RLT, QIAshredder spin column and EZ1 RNA Cell Mini Kit were purchased form Qiagen (Valencia, CA). The RNA 6000 Nano Reagent Kit was purchased from Agilent (Santa Clara, CA). The Affymetrix GeneChip 3′ IVT Express Kit was purchased from Affymetrix (Santa Clara, CA).