Transcriptor first strand cdna synthesis kit

Total mRNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNA was then synthesized using 2 μg of mRNA and a Transcriptor first-strand cDNA synthesis kit (Promega). Real-time qPCR was then performed as previously described [55 (link)]. β-actin was used for qPCR normalization, and all experiments were measured in triplicate. Primer sequences (5′-3′) are as follows:
Mouse-Ift20-Forward 5′-AGAAGCAGAGAACGAGAAGATG-3′
Mouse-Ift20-Reverse 5′-CACAAAGCTTCATATTCAACCCG-3′
Mouse-Ift88-Forward 5′-TGAGGACGACCTTTACTCTGG-3′
Mouse-Ift88-Reverse 5′-CTGCCATGACTGGTTCTCACT-3′
Mouse-Kif3a-Forward 5′-ATGCCGATCAATAAGTCGGAGA -3′
Mouse-Kif3a-Reverse 5′-GTTCCCCTCATTTCATCCACG-3′
Mouse-Ptch1-Forward 5′-CCGTTCAGCTCCGCACAGA-3′
Mouse-Ptch1-Reverse 5′-CTCACTCGGGTGGTCCCATAAA-3′
Mouse-β-actin-Forward 5′-AAGGCCAACCGTGAAAAGAT-3′
Mouse-β-actin-Reverse 5’-GTGGTACGACCAGAGGCATAC-3’
Human-IFT20-Forward 5′-GCAGCAACTTCAAGCCCTAAT-3′
Human-IFT20-Reverse 5′-ACGCCACCTCTTGTGACATAG-3′
Human-IFT88-Forward 5′-GCCGAAGCACTTAACACTTAT-3′
Human-IFT88-Reverse 5′-GTCTAATGCCATTCGGTAGAA-3′
Human-KIF3a-Forward 5′-GAGGAGAGTCTGCGTCAGTCT-3′
Human-KIF3a-Reverse 5′-CAGGCTTTGCAGAACGCTTTC-3′
Human-PTCH1-Forward 5′-CCAGAAAGTATATGCACTGGCA-3′
Human-PTCH1-Reverse 5′-GTGCTCGTACATTTGCTTGGG-3′
Human-β-actin-Forward 5′-CCCAGAGCAAGAGAGG-3′
Human-β-actin-Reverse 5′-GTCCAGACGCAGGATG-3′

Total mRNA in each group was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. Then, cDNA was synthesized using 2 μg of mRNA and a Transcriptor first‐strand cDNA synthesis kit (Promega). Real‐time qPCR was then performed as previously described.19, 20, 21 β‐actin was used for qPCR normalization, and all experiments were measured in triplicate. Primer sequences (5′‐3′) are as follows:
p62‐ Forward 5′‐GACTACGACTTGTGTAGCGTC‐3′.
p62‐ Reverse 5′‐AGTGTCCGTGTTTCACCTTCC‐3′.
β‐actin‐ Forward 5′‐CCCAGAGCAAGAGAGG‐3′.
β‐actin‐ Reverse 5′ ‐GTCCAGACGCAGGATG‐3′.

Total RNA was isolated from treated chondrocytes by using TRIzol solution (Thermo Fisher Scientific, MA, USA), and 2 μg of the total RNA from different samples was used for cDNA synthesis by using a Transcriptor First-Strand cDNA Synthesis Kit (Promega, WI, USA). Subsequently, PCR was performed using SYBR Green Master Mix (Takara, Tokyo, Japan). GAPDH gene expression was measured for normalization, and the 2^−ΔΔt method was used to calculate the relative expression level of target genes [22 (link)].

Real-time quantitative polymerase chain reaction was used to determine relative mRNA expression levels of tight junction protein ZO-1, claudin-1, occludin, and Muc-2. Colon tissue RNA levels were obtained and quantified using the Total RNA kit (Vazyme, Nanjing, China), and 2000C Ultra-micro UV spectrophotometer (Thermo Fisher Scientific Inc., USA), respectively. We used the Transcriptor First Strand cDNA Synthesis kit (Promega, Madison, USA) to synthesize cDNA for this study. The GoTaq°R SYBR-Green qPCR Master Mix (Promega, Madison, USA) was used to perform RT-qPCR corrections. The relative mRNA expressions of specific genes were calculated by the 2^−ΔΔCT method. GAPDH genes are used as internal reference genes. Supplementary Table S3 displays the specific gene primers as designed by Sangon Biotech Co., Ltd (Shanghai, China).

The total RNA was extracted using TRIzol solution (Thermo Fisher Scientific). The cDNA of different samples was synthesized using 2 μg of total RNA as well as the Transcriptor first-strand cDNA synthesis kit (Promega). Then the qRT-PCR was performed with SYBR Green Master Mix (TAKARA). The sequences of different primers are as follows (5′ to 3′):
Mouse Hepcidin -F 5CTGCGCCTTTTCAAGGATGG.
Mouse Hepcidin-R AATTGTTACAGCATTTACAGCAGAAGA.
Mouse Ptgs2-F CTGCGCCTTTTCAAGGATGG.
Mouse Ptgs2-R GGGGATACACCTCTCCACCA.
Mouse Actb-F AAATCGTGCGTGACATCAAAGA.
Mouse Actb-R GCCATCTCCTGCTCGAAGTC.
Human HEPCIDIN-F CTGACCAGTGGCTCTGTTTTC.
Human HEPCIDIN-R GAAGTGGGTGTCTCGCCTC.
Human ACTB-F CCCAGAGCAAGAGAGG.
Human ACTB-R GTCCAGACGCAGGATG.

Total mRNA was extracted using TRIzol (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions. cDNA was then synthesized using 2 μg of mRNA and a Transcriptor first‐strand cDNA synthesis kit (Promega, Madison, WI, USA). Real‐time qPCR was then performed as previously described [14 (link)]. GAPDH was used for qPCR normalization, and all experiments were measured in triplicate. Primer sequences (5’ to 3’) are as follows:
CHML F – AGGTTTGCCCGAATCCATCC
CHML R – TTCATGGATCAGGTCCTGCC
MTOR F – GTCTCGGCAACTTGACCATC
MTOR R – AAATGCTGCATGTGCTGGAA
S6KB1 F – CGACAGCCCAGATGACTCAA
S6KB1 R – ATTTGACTGGGCTGACAGGT
S6 F – TTGAAGTGGACGATGAACGC
S6 R – TTGTTTGTCGTTCCCACCAC
GADPH F – CTGACTTCAACAGCGACACC
GADPH R – TGCTGTAGCCAAATTCGTTGT

Total RNA was extracted from PBMCs using the Redzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. The extracted RNA was reverse-transcribed to first-strand cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Promega, Madison, WI, USA). Gene expression was quantified on an ABI 7500 thermal cycler (Applied Biosystems, Waltham, MA, USA) using the SYBR Green PCR Master Mix Kit (Promega). The amplification program was as follows: initial denaturation at 94°C for 5 min, followed by 40 cycles at 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, and 83°C for 1 min. β-Actin was used as internal control. The primer sequences used are shown in Table 1. For each sample, PCR was performed twice with triplicates for each sample, and the data were analyzed using the thermal cycler software to calculate the ΔCt value.