Kapa sybr fast qpcr

Blood samples were centrifuged at 2000 rpm for 5 min, and the RNA was extracted in 800 μl of the precipitate with TRIzol™ reagent (Invitrogen, Thermo Fisher Scientific), according to the manufacturer instructions. Extracted RNA was treated with TURBO DNA-free™ kit (Ambion^®, Life Technologies) and its concentration was determined by spectrophotometry (Epoch^®, Biotek Instruments). Relative gene expression was determined by real time RT-PCR assay based on SYBR-Green detection. TUBB was used as gene for normalization^{17 (link)}. cDNAs were obtained from 2 µg RNA using M-MLV Reverse Transcriptase (Promega^®) and random primers (Promega^®), according to the manufacturer’s guidelines. Primers to IL7R mRNA were designed using the Genbank sequence NM_002185.4 and the software Beacon Designer 8.02 (PREMIER Biosoft^®) (F: 5’-CAGCAATGTATGAGATTA-3’; R: 5’-ATGGTTAGTAAGATAGGAT-3’). The PCR reaction was performed using the KAPA SYBR^© Fast qPCR (Kapa Biosystems^®) with cDNA (2 µl) in a final volume (10 µl), according to the Eco™ Real-Time PCR system (Illumina^®) protocol. PCR amplification was performed at 95 °C for 3 min, and 45 cycles: 95 °C for 3 s, 53 °C for 15 s and 72 °C for 3 s followed by the melting curve (55–95 °C). Gene expression fold changes (FC) were determined using the 2^−ΔΔCTmethod^{18 (link)}. The group used as control is indicated in each analysis.

The protocol described in Section 2.2 was performed and total RNA was extracted by Trizol (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. RNA was treated with DNase I (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and then quantified. Reverse transcription was performed using 1 mg of total RNA and random primers (Thermo Scientific, Thermo Fisher Scientific, Waltham, MA, USA) in 20 μL of reaction mixture using the reverse transcription reagents kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). For PCR, a KAPA SYBR FAST qPCR (KAPABIOSYSTEMS, Sigma-Aldrich, Merck Bulgaria EAD, Bulgaria) kit was used. Reactions were made in triplicate using 2 μL of cDNA and a 0.4 μM final concentration of each primer in a 25 μL final volume. The following primers were used: β-actin, sense (5′-GATCTGGCACCACACCTTCT-3′) and antisense (5′-GGGGTGTTGAAGGTCTCAAA-3′); mlamp2a, sense (5′-AGGTGCTTTCTGTGTCTAGACCGT-3′) and antisense (5′-AGA ATA AGT ACT CCT CCC AGA GCT GC-3′). Reactions were subjected to dissociation curve analysis to exclude the possibility of nonspecific amplification. Changes for each gene were calculated using the mean of the change in the Ct values (ΔCt) normalized to the Ct values of β-actin for each sample (2^−ΔΔCt).

The retrotranscription reaction was performed with 1 μg of polysomal or total RNA using the iScriptcDNA synthesis kit (Biorad) in accordance with the manufacturer’s instructions. The obtained cDNA was used as template in aqPCR reaction with the KAPA SYBR FAST qPCR (Kapa Biosystem) and specific primers as reported in Table S3. qPCR were run in three biological and three technical replicates. The relative expression was calculated with the delta delta Ct method. Gapdh and Als2 were used as reference genes. The gene-specific Translation Efficiency (TE) was calculated as the ratio between the fold change at the polysomal level and the fold change at the total level of the gene of interest.

Total RNA was extracted by using Trizol (Invitrogen) according to the manufacturer’s instructions, treated with DNase I (Invitrogen) and then quantified. Reverse transcription was performed using 1 μg of total RNA and random primers (Thermo Scientific) in 20 μl of the total volume of the reaction mixture using the reverse transcription reagents kit (Invitrogen). For PCR, KAPA SYBR FAST qPCR (KAPABIOSYSTEMS) kit was used. Reactions were made in triplicate using 2 μl of cDNA and 0.4 μM final concentration of each primer in 25 μl final volume. Primers were the following: β-actin, sense (5′-GATCTGGCACCACACCTTCT-3′) and antisense (5′-GGGGTGTTGAAGGTCTCAAA-3′); hHsc70, sense (5′-GGAGGTGGCACTTTTGATGT-3′) and antisense (5′-AGCAGTACGGAGGCGTCTTA-3′); hLamp2A, sense (5′-TATGTGCAACAAAGAGCAGA-3′) and antisense (5′-CAGCATGATGGTGCTTGAGA-3′). Reactions were subjected to dissociation curve analysis to exclude the possibility of nonspecific amplification. Changes for each gene were calculated using the mean of the change in Ct values (ΔCt) normalized to the Ct values of β-actin for each sample (2^−ΔΔCt).

DNA was extracted from fecal samples (n = 144) using the DNeasy PowerSoil kit (Qiagen, Germantown, MD). DNA yield and purity were evaluated on a Nanodrop (Life Technologies Corp., Grand Island, NY) and by electrophoresis on a 0.8% agarose gel. Previously described primers and conditions were used to amplify and sequence the V4 region of the 16S rRNA gene [31 (link)]. Each PCR mixture contained 17 μl AccuPrime Pfx SuperMix (Life Technologies Corp., Grand Island, NY), 5.0 μM each of the primers, and 25 ng of the template DNA. PCR settings included denaturation at 95°C for 2 min and 22 cycles of (20 seconds at 95°C, 15 seconds at 55°C, 5 min 72°C) amplification followed by final extension at 72°C for 10 min. PCR amplicons were normalized using the SequalPrep^™ Normalization Plate (96) Kit (Applied Biosystems Inc., Foster City, CA). Normalized amplicons were pooled and quantified using Kapa SYBR Fast qPCR (Kapa Biosystems, Wilmington, MA) and sequenced on a MiSeq Instrument using a MiSeq Reagent Kit v2 following manufacturer’s instructions (Illumina, San Diego, CA). DNA from a mock community with defined composition [32 (link)] was also used to calculate sequencing error rates.

Total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany) and cDNA was synthesized from 1 μg of total RNA with random hexamers using the RevertAid First Strand cDNA Synthesis Kit (Thermo) according to the manufacturer’s instructions. Quantitative RT–PCR was performed using Kapa Sybr Fast qPCR (Kapa Biosystems, Wilmington, MA, USA) on the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). PCR reactions were carried out in biological triplicates and technical triplicates (n = 9) and relative expression was calculated using the comparative CT method with 18S ribosomal RNA expression as the reference control. See Supplementary Materials for primer sequences.