strain: Sprague Dawley gender: Male age: 9 weeks old treatment: saline vehicle given to neonate rat and TNBS given as adult tissue: Colonic Mucosa/Submucosa
Treatment protocol
The rats were divided into four treatment groups delivered intraluminally into the colon given a combination of vehicle (Ctl; normal saline) and/or trinitrobenzene sulfonic acid (TNBS) (NI/AI). The four groups as previously described were Ctl+Ctl, Ctl+AI, NI+Ctl, and NI+AI. To induce neonatal inflammation, TNBS (130 mg/kg dissolved in 200 µl saline containing 10% ethanol) was injected intraluminally 2 cm into the colon of male pups on post-natal day 10. The animals were kept in a head-down position while the anus was held closed for one minute to prevent leakage. Rats in the sham treatment groups received 200 µl of saline. Six to eight weeks later, animals were subjected to a secondary TNBS (65 mg/kg) insult, as AI. Under light anesthesia, 250 µl of TNBS in PBS containing 40% ethanol was injected intrarectally via a catheter, advanced to 8 cm into the colon. Control rats were given 250 μl of saline. Seven days after the second TNBS treatment, animals were decapitated under anesthesia. The colon was flushed clean with PBS and cut open longitudinally. The colonic mucosa and submucosa were isolated, snap-frozen in liquid nitrogen, pulverized, aliquoted, and stored in a -80 °C freezer.
Extracted molecule
total RNA
Extraction protocol
Total RNA containing miRNA was extracted from the pulverized colonic mucosa/submucosa using the miRNeasy Mini Kit (QIAGEN, Valencia, CA), according to the protocol provided by the manufacturer. RNA concentration was quantified in a Nanodrop. After quantification, RNA was stored in a -80 °C freezer.
Label
Cy5
Label protocol
Microarray assay was performed using a service provider (LC Sciences). Total RNA sample (2 µg) were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies) (1). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://mirbase.org) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. (1) (a) Gao, X., Gulari, E., and Zhou, X. (2004) In situ synthesis of oligonucleotide microarrays. Biopolymers 73, 579-596; (b) Zhu, Q., Hong, A., Sheng, N., Zhang, X., Jun, K.-Y., Srivannavit, O., Gulari, E., Gao, X., and Zhou, X. (2007) Microfluidic biochip for nucleic acid and protein analysis. in Methods Mol. Biol. Ed. Rampal, J. B. 382:287-312.
Hybridization protocol
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://miRBase.org) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.
Scan protocol
The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 mL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C. After RNA hybridization, tag-conjugating Alexa Fluor®546 dye was circulated through the microfluidic chip for dye staining. Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
Description
Ctl+AI
Data processing
Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression) (2). (2) Bolstad, B. M., Irizarry, R. A., Astrandand, M., Speed, T. P. (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinfo. 19, 185-193.
