One step sybr reverse transcription pcr rt pcr kit

Jejunum tissue was collected from mice sacrificed either before or after the challenge with OVA. Total RNA was extracted with phenol. A 50 ng aliquot of total RNA was reverse-transcribed and amplified with a One-Step SYBR reverse transcription PCR (RT-PCR) kit (TaKaRa Bio Inc, Shiga, Japan) in the PikoReal Real-Time PCR System (Thermo Fisher Scientific, Inc., Kanagawa, Japan). The specific primer pairs were designed based on published data for 18S ribosomal RNA (rRNA), Ocln, Cldn3, and E4bp4 genes40 (link)41 (link)42 (link). The primer sequences are described in Supplementary Table. S1. The relative level of the target gene PCR product was normalized to that of 18s rRNA. The data were analyzed using the ΔΔCt method.

Total RNA was extracted from tissues, using phenol. Aliquots of 50 ng of total RNA were reverse-transcribed and amplified using a One-Step SYBR reverse transcription PCR (RT-PCR) kit (TaKaRa Bio, Shiga, Japan) in Piko Real (Thermo Fisher Scientific, Tokyo, Japan). Primer pairs were designed using OligoAnalyzer software (Integrated DNA Technologies, Singapore) and the primers used in this study are listed in Table 1. The relative level of target gene was calculated using 2^−ΔΔCT method using 18srrna as a reference gene and normalized with untreated control.

Total RNA was extracted from tissues, using phenol. Aliquots of 50 ng of total RNA were reverse-transcribed and amplified using a One-Step SYBR reverse transcription PCR (RT-PCR) kit (TaKaRa Bio Inc, Shiga, Japan) in Piko Real (Thermo Fisher Scientific Inc, Kanagawa, Japan). Primer pairs were designed based on published data for the Gapdh, Per1, Per2, and Pigr genes. The relative levels of the target gene PCR products were normalized to that of Gapdh. The data were analyzed using the delta-delta Ct method. The primers for Gapdh were as follows: Gapdh-F, 5′-TGGTGAAGGTCGGTGTGAAC-3′; Gapdh-R, 5′-AATGAAGGGGTCGTTGATGG-3′. The primers for Per1 were as follows: Per1-F, 5′-CAAGTGGCAATGAGTCCAACG-3′; Per1-R, 5′-CGAAGTTTGAGCTCCCGAAGTG-3′. The primers for Per2 were as follows: Per2-F, 5′-TGTGTGCTTACACGGGTGTCCTA-3′; Per2-R, 5′-ACGTTTGGTTTGCGCATGAA-3′. The primers for pIgR were as follows: pIgR-F, 5′-AGTAACCGAGGCCTGTCCT-3′; pIgR-R, 5′-GTCACTCGGCAACTCAGGA-3′.