Mmlv reverse transcription kit

The RNeasy Mini Kit (Qiagen) was used to extract RNA. cDNA was synthesized from 500 ng RNA with the MMLV Reverse Transcription Kit (Evrogen, Moscow, Russia) according to the manufacturer’s instructions. The relative expression of gene-markers adipogenic differentiation PPARγ and adiponectin was analyzed by quantitative real-time PCR. The following equipment were used: qPCR mix-HS SYBR + LowROX (Evrogen) reagents and CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States). The gene of 60S Ribosomal protein P0 (RPLP0) was used as a housekeeping gene. Quantification and normalization of expression levels of the target genes and the reference gene (RPLP0) were calculated using the comparative threshold cycle (CT) method. Primers for PCR were picked using the NCBI Primer Designing Tool. Primer sequences are presented below:
60S Ribosomal protein P0 (RPLP0), For: GCT​GCT​GCC​CGT​GCT​GGT​G, Rev: TGG​TGC​CCC​TGG​AGA​TTT​TAG​TGG, 130bp. Adiponectin (ADIPOQ), For: GAC​CAG​GAA​ACC​ACG​ACT​CA, Rev: TTT​CAC​CGA​TGT​CTC​CCT​TAG​G, 199bp. Peroxisome proliferator-activated receptor gamma (PPARγ), For: TCA​GGT​TTG​GGC​GGA​TGC, Rev: TCA​GCG​GGA​AGG​ACT​TTA​TGT​ATG, 147bp.

Viral RNA Mini Kit was from QIAGEN, Hilden, Germany. MMLV Reverse Transcription kit, random primers, nuclease free water, DNA ladder, and TAE buffer were from Evrogen, Moscow, Russia. MycoKill AB solution was from PAA Laboratories GmbH, Pasching, Oberosterreich, Austria.

RNA was isolated using the ExtractRNA kit (Evrogen, Moscow, Russia). RNA concentration was determined using a Nanodrop One C spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using the MMLV reverse transcription kit (Evrogen) according to the manufacturer’s protocol. qPCR was performed on a LightCycler 96 (Roche, Basel, Switzerland) with qPCRmix-HS SYBR reagent (Evrogen). The cycling conditions were 95 °C for 150 s, followed by 45 cycles of 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s. Data were collected using LightCycler software (version 4.1). The 18S RNA was used as an intermediate control. The primer sequences are provided in Supplementary Table S1.

Fetuin and horseradish peroxidase were from Serva, Switzerland. Antibodies against mouse and chicken immunoglobulins conjugated with horseradish peroxidase were from Sigma-Aldrich, Inc., St. Louis, MO, USA. MycoKill AB solutions were from PAA Laboratories GmbH, Pasching, Austria. Viral RNA Mini Kit was from QIAGEN, Hilden, Germany. MMLV Reverse Transcription kit, random primers, nuclease-free water, DNA Ladder, and TAE buffer were from Evrogen, Moscow, Russia. Ribonuclease inhibitors were from Syntol, Moscow, Russia. Soluble synthetic polyN-(2-hydroxyethyl)acrylamide-based sialylglycopolymers (SGP) contained 20 mol% of specific sialyloligosaccharide attached to the 30-kDa polymer were from GlycoNZ, Auckland, New Zealand.

Total RNA was isolated using the Total RNA Purification Kit (Jena Biosciences) according to the manufacturer’s protocol. Residual genomic DNA was removed using DNase I (Thermo Fisher Scientific, Walthon, MA USA) according to the manufacturer’s protocol; 1 U of the enzyme was used per RNA sample. cDNA was synthesized using the MMLV reverse transcription kit (Evrogen, Moscow, Russia) with an oligo-dT primer. PCR was performed using the DreamTaq master mix (Thermo Fisher Scientific, Walthon, MA USA); the program was as follows. Initial denaturation at 95 °C for 3 min, cycle: denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, DNA synthesis at 72 °C for 30 s for 35 cycles, final DNA synthesis at 72 °C for 5 min. The primers were generated using the ITDNA PrimerQuest tool (https://eu.idtdna.com/PrimerQuest; accessed on 1 January 2020) and validated using the NCBI Primer-BLAST service [38 (link)].

RNA was extracted using the RNEasy mini kit (Qiagen) with the addition of the Plant RNA Isolation Aid solution (Ambion) to the lysis buffer. RNA was treated twice with RNase-free DNAse (Qiagen). cDNA synthesis was performed using the MMLV reverse transcription kit (Evrogen). Following reverse transcription, PCR was performed using primers Pyrrot-rps4-F (5′-GGGAGCTTTACCAGGACTAAC-3′) and Pyrrot-rps4-div-R (5′-AATGTTAGTGGACGGTGGTATC-3′) for rps4-like ORF357. As a positive control, we also amplified two photosynthesis-related genes – petB (Pyrrot-petB-F 5′-ATGAGTAAAGTCTACGATTGGTTC-3′ and Pyrrot-petB-R 5′-AAACGAGTCAAAGTGGATTGTC-3′) and psaB (Pyrrot-psaB-F-5′-ACTCGTCGTATTTGGTTTGGGATT-3′ and Pyrrot-psaB-R 5′-TTATTCCATCGGACAAACTCTCC-3′). A negative control with no reverse transcriptase added was also included.

Fetuin and horseradish peroxidase were from Serva, Oftringen, Switzerland. Antibodies against mouse immunoglobulins conjugated with horseradish peroxidase were from Sigma, St. Louis, MO, USA. Viral RNA Mini Kit was from QIAGEN, Hilden, Germany. MycoKill AB solution was from PAA Laboratories GmbH, Pasching, Austria. Ethidium Bromide Solution was from Promega, Madison, WI, USA. MMLV Reverse Transcription kit, random primers, nuclease free water, DNA Ladder, and TAE buffer were from Evrogen, Moscow, Russia. Ribonuclease Inhibitor was from Syntol, Moscow, Russia. Embryonated chicken eggs were purchased from the State poultry farm “Ptichnoe” (Moscow, Russia).