Mmlv reverse transcriptase kit

Total RNA isolated from mouse intestines (24h post-IR) (n = 3 per group and one sample per mouse) was subjected to reverse transcription with Superscript II RNase H—Reverse Transcriptase and random hexanucleotide primers. The RNA samples were assessed for quantification and purity by A260/A280 absorption, and RNA samples with ratios greater than 1.8 were stored at −80°C for further analysis. qRT-PCR was performed using a MMLV-Reverse Transcriptase kit (EPICENTRE® Biotechnologies, Madison, WI, USA). Complementary DNA (cDNA) was then synthesized. Information for primers used in qRT-PCR was described in Supplementary Table 1.

Total RNA was extracted from the cultured cells using TRIzol reagent (Invitrogen), and then quantified using an Epoch Microplate Spectrophotometer with a Take3 Micro‐Volume Plate (BioTek, Winooski, VT). One microgram of total RNA was reverse‐transcribed using an MMLV reverse transcriptase kit with random primers (Epicentre Biotechnologies, Madison, WI). The specific primers for real‐time quantitative PCR were designed by qPrimerDepot (http://primerdepot.nci.nih.gov/) (Table 1). The real‐time PCR reactions were performed in 20 μL volumes containing 10 μL of Real‐time PCR DyNAmo Flash SYBR Green qPCR reagent (Thermo Fisher Scientific Inc., NYSE: TMO). The PCR reaction was performed using a PikoReal™ 96 Real‐Time PCR System (Thermo Fisher Scientific Inc.), and the conditions were as follows: 45 cycles of 95°C for 5 sec and 60°C for 1 min. The data were analyzed using PiKoReal Software 2.0, exported into Excel for analysis using the ΔC_t (the number of PCR cycles to reach the threshold of the product detection) method, and normalized to β‐actin as an internal control. The quantitative real‐time PCR experiments adhered to the MIQE (Minimum Information for Publication of Quantitative Real‐Time PCR Experiments) guidelines (http://www.clinchem.org/cgi/content/short/55/4/611).

We used qPCR to validate the differentially expressed miRNAs identified in the validation study of miRNA arrays. Total RNA extracted from PBMCs was used to synthesize cDNA using miRNA-specific, stem-loop reverse transcription (RT) primers and reagents from a MMLV Reverse Transcriptase kit (Epicentre, Madison, USA), following the manufacturer’s protocol. Next, we performed qPCR with ViiA 7 Real-time PCR System (Applied Biosystems, Foster, USA), and 2×SYBR-Green PCR master mix (Arraystar, Rockville, USA) according to the manufacturer’s instructions. We used the following primers: hsa-miR-223-3p F: 5’GGGGTGTCAGTTTGTCAAA3’, R:5’CAGTGCGTGTCGTGGAGT3’. hsa-miR-21-5p F: 5' GGGGGGTAGCTTATC AGACTG3', R:5'CAGTGC GTGTCGTGGAGT3'. All reactions were performed in triplicate. The expression level of each miRNA was calculated relative to U6 RNA. The relative expression level of each miRNA was calculated using the equation 2^-ΔΔCt.