Total RNA was extracted from 50 mg sWAT using Trizol reagent (Invitrogen, CA, USA). RNA quantity was determined using Nanovue (GE Life Sciences) spectroscopy, and 1 mg RNA was treated with DNAse I (Invitrogen). First-strand cDNA synthesis was performed using Oligo (dT) primers for mRNA and Superscript III reverse-transcriptase (both Invitrogen). Quantitative real-time PCR (RT-qPCR) was performed using a StepOne plus cycler and the SYBR Green mix (Invitrogen). Primers were designed using Primer3web online software version 4.0.0 and are indicated in Table 1 . The beta-actin gene was used as an endogenous control to normalize selected gene expression. Efficiencies of RT-qPCR for the target gene and the endogenous control were approximately equal and were calculated from a cDNA dilution series. Real-time PCR reactions were conducted as previously detailed [12 (link)]. Negative controls consisted of wells in which cDNA was substituted for deionized water. The relative mRNA expression ratio (RQ) was calculated using the 2-∆∆ct equation in which 2∆CT expresses the difference between the number of cycles (CT) of the target genes and the endogenous control.
Oligo dt primers for mrna
Manufactured by Thermo Fisher Scientific
Oligo (dT) primers are short synthetic DNA sequences that are designed to specifically bind to the poly(A) tail of mRNA molecules. They serve as primers for the reverse transcription of mRNA into complementary DNA (cDNA), which is a critical step in various molecular biology techniques, such as gene expression analysis and cDNA library construction.
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Lab products found in correlation
Sybr green mix, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Steponeplus cycler, by Thermo Fisher Scientific (1 mentions)
Nanovue, by GE Healthcare (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cfx96 thermocycler, by Bio-Rad (1 mentions)
2 protocols using oligo dt primers for mrna
1
Real-Time PCR Analysis of Gene Expression
2
Mitochondrial Biogenesis Markers Quantification
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We extracted total RNA with TRIzol (Invitrogen, Carlsbad, CA) in 30 mg of the liver, eWAT, and gastrocnemius muscle (quantified with Nanovue spectroscopy, GE Healthcare Biosciences, Pittsburgh, PA) (n ϭ 6/group, the three tissues and all primers used). Then, 1 g of RNA was treated with DNase I (Invitrogen). First-strand cDNA was synthesized using Oligo (dT) primers for mRNA and Superscript III reverse transcriptase (both Invitrogen). Quantitative real-time PCR was performed using a CFX96 thermocycler (Bio-Rad, Hercules, CA) and SYBR Green mix (Invitrogen). Primers are demonstrated in Table 2, and their design followed the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines (7) (link). We evaluated the gene expression of the following markers: 1) peroxisome proliferator-activated receptor gamma coactivator 1-␣ (PGC1␣), 2) peroxisome proliferator-activated receptor gamma coactivator 1- (PGC1), 3) fibronectin type III domain containing 5 (FNDC5), 4) nuclear respiratory factor 1 (NRF1), 5) mitochondrial transcription factor A (TFAM), 6) peroxisome proliferator-activated receptor ␣ (PPAR␣), and 7) carnitine palmitoyltransferase 1 (CPT1).
We standardized the expression of the selected genes with the endogenous expressions of -actin and TATA-box protein (TBP).
We standardized the expression of the selected genes with the endogenous expressions of -actin and TATA-box protein (TBP).
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