Mini rna isolation iitm

At week 16, the mice were sacrificed and the epididymal fat pads were dissected. RNA extraction was performed using a Mini RNA Isolation IITM (Zymo Research, Orange, CA, USA). RNA from liver tissue was extracted using Trizol reagent. To evaluate the gene expression levels, we performed quantitative real-time polymerase chain reaction (qRT-PCR). The complementary DNA (cDNA) was synthesized using an Advantage RT for PCR Kit (Clontech, Palo Alto, CA, USA). The sequences of primes used in this study are shown in Table 1. PCR was carried out in a 7900HT Fast Real-Time PCR System (Applied Biosystems®, Foster City, CA, USA). For gene expression analysis, threshold cycle (Ct) of each gene was calculated by SDS Software 2.4 (Applied Biosystems, Foster City, CA, USA); then relative quantitation (RQ) was performed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, housekeeping gene). The fold change was calculated according to NC group which was considered as 1.

At week 4, the rats were sacrificed and the livers were dissected. RNA extraction was performed using a Mini RNA Isolation IITM (Zymo Research Corp, CA, USA). RNA was extracted using TRIzol reagent. To evaluate gene expression including deiodinase 1 (Dio1), thyroid hormone responsive spot 14 (Thrsp or Spot14), and thyroxine-binding globulin (Tbg), quantitative real-time polymerase chain reaction (qRT-PCR) was performed. Prior to qRT-PCR, the complementary DNA (cDNA) was synthesized using an Advantage RT for PCR Kit (Clontech, USA). To the cDNA obtained through reverse transcription PCR, 2x SYBR reaction buffer, primers, and dH₂O were added, and qRT-PCR was carried out using 7900HT Fast Real-Time PCR System (Applied Biosystems®, USA). The primer sequencing is as follows: Dio1, 5′-TTTAAGAACAACGTGGACATCAGG-3′ and 5′-GGTTTACCCTTGTAGCAGATCCT-3′; Spot14, 5′-CTTACCCACCTGACCCAGAA-3′ and 5′-CATCGTCTTCCCTCTCGTGT-3′; Tbg, 5′-GCTGCTTTAGCCATGCTTTC-3′ and 5′-AAACTGCATTTCCCATCTGC-3′; and GAPDH, 5′-GTCGGTGTCAACGGATTTG-3′ and 5′-AGCTTCCCATTCTCAGCC-3′. For gene expression analysis, the threshold cycle for each gene, obtained with SDS Software 2.4 (Applied Biosystems®, USA), was converted to relative quantitation based on GAPDH, and the fold change was calculated. The fold change value of each experimental group was normalized according to the Normal group, which was defined as 1.

At week 17, the mice were sacrificed and livers were dissected. RNA extraction was performed using a Mini RNA Isolation IITM (Zymo Research Corp, Irvine, CA, USA). RNA was extracted using TRIzol reagent. To evaluate gene expression including TNF-α, interferon gamma (IFN-γ), and F4/80, quantitative real time-polymerase chain reaction (qRT-PCR) was performed. Prior to qRT-PCR, the complementary DNA (cDNA) was synthesized using an Advantage RT for PCR Kit (Clontech, Mountain View, CA, USA). To the cDNA obtained through reverse transcription PCR, 2× SYBR Reaction buffer, primers, and dH₂O were added, and qRT-PCR was carried out using 7900HT Fast Real-Time PCR System (Applied Biosystems^®, Waltham, MA, USA). For gene expression analysis, the threshold cycle for each gene, obtained with SDS Software 2.4 (Applied Biosystems^®, Waltham, MA, USA), was converted to relative quantitation based on GAPDH, and the fold change was calculated. The fold change value of each experimental group was normalized according to the NC group, which was defined as 1.