Rna bee rna isolation solvent

To measure the levels of cytokine transcripts, the rolled colon was snap frozen and subjected to total RNA extraction and quantitative reverse transcription polymerase chain reaction for IL-1β, IL-17, IL-6 and TNF-α. Total RNA was isolated with RNA-Bee RNA Isolation solvent (TEL-TEST, Inc., Friendswood, TX). After the RNA was treated with DNase I, double-stranded cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Expression levels were quantified with a TaqMan gene expression kit for TNF-α, IL-17, TGFβ1 and 18s rRNA (TaqMan Gene expression assay ID was NM_013693.2, Mm00439618_m1, Mm01178820_m1 and Hs99999901_s1, respectively) or a SYBR Green PCR Master Mix (for IL-6 and IL-1β) using an ABI Prism^® 7900HT Sequence Detection System. Primers used were as follows: mouse IL-6, 5'-TGGAGTCACAGAAGGAGTGGCT AAG and 5'-TCTGACCACAGTGAGGAATGTCCAC; and IL-1β, 5'-CAACCAACAAGTGATATTCTC and 5'-GATCCACAC TCTCCAGCTGCA. Threshold cycle numbers (Ct) were determined with Sequence Detector Software (ver. 1.7; Applied Biosystems) and transformed using the ΔCt/ΔΔCt method as described by the manufacturer, with 18s rRNA used as endogenous control.

Total RNA was extracted using the RNA-Bee RNA isolation solvent (Tel-Test); 5 μg RNA was then converted into cDNA using the Moloney murine leukemia virus reverse transcriptase (Promega). Real-time PCR primers targeting human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and bFGF were designed using the Primer Express software (Applied Biosystems); the sequences are presented in Supplementary Table S1. The ABI Prism 7500 Sequence Detection System and the SYBR Green Master Mix kit (both from Applied Biosystems) were used for the real-time PCR analysis of the reverse-transcribed cDNA samples. The expression level of human GAPDH was used as an internal reference. Relative gene expression levels were calculated with the 2^-ΔΔCT method [14 (link)].