Mulv reverse transcriptase

cDNA synthesis was essentially performed as previously described
[10 (link)]. Briefly, single tube poly (A) synthesis and reverse transcription was performed with 100 ng of RNA in a final volume of 10 μl combined with 1 μl MuLV reverse transcriptase (New England Biolabs), 1 μl of poly (A) polymerase (New England Biolabs), 1 μl 10x poly (A) polymerase buffer, 1 μl 0.1 mM ATP, 1 μl 10 μM RT-primer (5′-CAGGTCCAGTTTTTTTTTTTTTTTVN where V is A, C and G and N is A, C, G and T). The reaction was incubated at 42°C for 1 hour followed by enzyme inactivation at 95°C for 5 minutes. A panel of three technical cDNA replicates for each of the three biological samples per condition was constructed. The cDNA was diluted 5× before used in qPCR reactions.
miR-specific qPCR was performed as previously described in
[16 (link)]. Briefly, 0.25 μl of 10 μM miRNA specific forward and 0.25 μl of 10 μM miRNA specific reverse primer, 5 μl of 2× LightCycler 480 SYBRGreen I Mastermix (Roche) and 3.5 μl of RNAse free H₂O and 1 μl of cDNA of each samples was combined in 10 μl reaction and subjected to qPCR performed on LightCycler 480 (Roche).

RNA was harvested using TRI reagent according to the manufacturer’s protocol. Following extraction, RNA was treated with RQ1 DNase (Promega). Random hexamers were used to prime cDNA synthesis with MuLV reverse transcriptase (New England Biolabs). Real-time reactions used iTaq Universal SYBR Green Supermix (Biorad). Primers are listed in S4 Table.

RNA was harvested using TriReagent according to the manufacturer’s protocol. Following extraction, RNA was treated with RQ1 DNase (Promega). Random hexamers were used to prime cDNA synthesis with MuLV reverse transcriptase (NEB). Real-time reactions used iTaq Universal SYBR Green Supermix (Biorad).

1 µg of RNA was reverse transcribed into cDNA in 50 µL reactions using the Taqman kit (Applied Biosystems), according to the manufacturer’s protocol. 1 μL of this reaction was used in subsequent qPCR reactions for the assessment of mRNA abundance, which were performed using the SYBR Green system (Applied Biosystems) on the ABI 7900 Fast Sequence Detection instrument, according to manufacturer’s specifications. All primer sequences are listed in Supplementary Table S1. mRNA expression was normalized to GAPDH (primer pair 4) and verified using beta actin (primer pair 5) and Beta-2 microglobulin (primer pair 6). miRNA RT-qPCR was performed as described previously (Busk 2011 BMC Biotechnology). Briefly, 500 ng of RNA was reverse transcribed into cDNA in 50 µL reactions containing 5 µL 10x poly(A) polymerase buffer, 0.1 mM ATP, 1 µM of RT primer (was 5′-CAGGTCCAGTTTTTTTTTTTTTTTVN, where V is A, C and G and N is A, C, G and T.), 0.1 mM of each deoxynucleotide (dATP, dCTP, dGTP, and dTTP), 500 units MuLV reverse transcriptase (New England Biolabs), and five units of poly(A) polymerase (New England Biolabs). The reaction was incubated for 1 h at 42°C, followed by enzyme inactivation at 95°C for 5 min qPCR was performed using the SYBR Green system as above. Snord 70 (primer pair 42) was used for normalization.

For mRNA and lncRNA, the Verso cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) was employed to synthesize cDNA for qRT-PCR from the RNA extracted above. It was carried out based on the manufacturer’s recommendations. Briefly, RNA (0.25–1 µg) was heated to 70 °C for 5 min and cooled to 4 °C before adding the anchored-oligo dT primer and other reagents supplied in the kit, including the RT enhancer that prevents genomic DNA carryover. The cDNA synthesis reaction was executed at 42 °C for 60 min, followed by 52 °C for 30 min, and lastly, enzyme inactivation at 95 °C for 2 min.
For miRNAs, cDNA was synthesized based on a published protocol [68 (link)]. Briefly, the 10 µL reaction, which included 1 µg RNA, 1 µL of 10X poly(A) polymerase buffer (New England Biolabs, Ipswich, MA, USA), 0.25 mM ATP, 1 mM dNTPs (New England Biolabs, USA), 1 µM RT-primer (GGTCCAGTTTT TTTTTTTTTTTVN (V is A, C, and G; N is A, C, G, and T)), 80 U MuLV reverse transcriptase (New England Biolabs, USA), and 0.75 U poly(A) polymerase (New England Biolabs, Ipswich, MA, USA) were subjected to 42 °C for 1 h, followed by enzyme inactivation at 95 °C for 5 min.

RNA was harvested with TRI reagent according to the manufacturer’s protocol. Purified RNA was treated with RQ1 DNase (Promega) and cDNA was synthesized with murine leukemia virus (MuLV) reverse transcriptase (New England Biolabs) with oligo(dT)20 priming. All real-time PCR mixes performed with iTaq Universal SYBR green Supermix (Bio-Rad) and analyzed with QuantStudio (v1.5.2). Gene-specific primer sequences can be found in Supplementary Table 1.

Total RNA was used for cDNA synthesis as previously described [34 ,35 (link)]. For subcutaneous adipose tissue the same RNA samples used for sequencing were used for qPCR. Briefly, for miRNA analysis, two cDNA replicates per sample were made from 100 ng of RNA in a final volume of 10 μl including 1 μl of 10x poly(A) polymerase buffer, 0.1 mM of ATP, 1 μM of RT-primer, 0.1 mM of each deoxynucleotide (dATP, dCTP, dGTP and dTTP), 100 units of MuLV reverse transcriptase (New England Biolabs, USA) and 1 unit of poly(A) polymerase (New England Biolabs, USA) were incubated at 42°C for 1 hour followed by enzyme inactivation at 95°C for 5 minutes. The sequence of the RT-primer was 5’-CAGGTCCAGTTTT TTTTTTTTTTTVN, where V is A, C and G and N is A, C, G and T. The primer was purchased from TAG Copenhagen (Denmark).
For mRNA analysis two cDNA replicates were made from 100 ng RNA for each sample in a final volume of 10 μl including 0.25 μg 3:1 mixture of random hexamers/OligodT, 2 μl Improm-II buffer, 2 mM dNTP mix, 10 units RNasin Ribonuclease inhibitor, 1.25 mM MgCl₂ and 0.5 μl Improm-II reverse transcriptase (Promega), which were incubated for 42°C for 1 hour and 15 min at 70°C following manufacturer’s instructions.