Lightcycler faststart dna master sybr green

Total RNA and DNA were simultaneously extracted [12] and reverse transcription (RT) reactions were done on 1 μg of total cell culture-extracted RNA or 100 ng of tissue-derived RNA with SuperscriptII (Invitrogen) and poly-dT primer. Quantitative PCR (qPCR) was performed on a LightCycler (Roche) using LightCycler Fast Start DNA Master SYBR Green (Roche). Primer sequences for qPCR reactions are listed in Table S1 in File S1. For all RT-PCR experiments the expression of target genes was normalized to the geometric mean of expression of three housekeeping genes [hypoxanthine phosphoribosyltransferase (Hprt), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and ATP synthase-coupling factor 6 (Atp5j)] to minimize variations due to different experimental conditions.

Total RNA was extracted from frozen tissues using TRIzol reagent (Invitrogen, USA). A Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific, Vilnius, Lithuania) was used to prepare reverse transcription according to the manufacturer’s protocols. qRT‒PCR was carried out using LightCycler-Fast-Start DNA Master SYBR Green (Roche Diagnostics, Tokyo, Japan). Gene expression was normalized to β-actin. The mRNA levels were expressed as the threshold cycle (CT). The amount of target was measured using the 2^-△△CT method. The primers used for qRT‒PCR were UBE2C-F: 5′-AGTGGCTACCCTTACAATGCG-3′ and UBE2C-R: 5′-TTACCCTGGGTGTCCACGTT-3′.

Extraction of total RNA from frozen tissues was performed using TRIzol reagent (Invitrogen, USA). RevertAid First-Strand cDNA Synthesis Kit (Thermoscientific, Vilnius, Lithuania) was used to prepare reverse transcription according to the manufacturer’s protocols. The qRT-PCR reaction was carried out using LightCycler-Fast-Start DNA Master SYBR Green (Roche Diagnostics, Tokyo, Japan). Gene expression was normalized to β-actin. Three parallel reactions were set for each sample. The mRNA expression value were calculated by the MxPro QPCR Software 4.10 (Mx3005P, Stratagene, Agilent Technologies) according to 2-^△△CT method. The primers used for qRT-PCR were: CHCHD2, F-5′- CAG TTG GCT CTTBCTGBCTG CT-3′ and R-5′-GTA ATG GCG TGA CCC AAT GT-3′; HIF-1α, F-5′-TGC AAC ATG GAA GGT ATT GC − 3′ and R-5′-TTC ACA AAT CAG CAC CAA GC − 3′; β-actin, F-5′-TCC CTG GAG AAG AGC TAC GA-3′ and R-5′-AGC ACT GTG TTG GCG TAC AG-3′.

RNA extraction and purification were as reported, using a minimum of three biological replicates per group.^{12–14 (link),84–86 (link)} After SuperScript III (Thermo Fisher Scientific) or miScript RT II (Qiagen) kits, quantitative PCR (qPCR) reactions were performed by LightCycler® FastStart DNA Master SYBR Green (Roche Applied Science) on a LightCycler96 instrument. Primers for mRNA qPCR were designed by NCBI-BLAST (see Supplemental Table S11), whereas primers for miRNA qPCR were purchased custom-made from Qiagen. Internal controls were Gapdh and U6B small nuclear RNA for mRNA and miRNA analyses, respectively. RNAs and miRNAs were selected based on prior validation and ranking in GSEA data. Original gene lists for tight junction and anti-microbial function were obtained from KEGG and Gene Ontology (GO) resources, respectively, and further sorted by the sequencing data to generate genes of interest. We focused on six miRNAs consistently altered in Pirc colon tumors, and filtered miRNA-mRNA pairs by conserved UTR target site in human and rat, with a linear correlation <-0.7 in sequencing data. Verification of miRNAs and mRNAs was by qPCR (Pearson’s test, with r <-0.5).

Total RNA was extracted via TRIzol reagent (Invitrogen, UK) according to the manufacturer's protocol. The first strands of complementary DNAs (cDNA) were synthesized by reverse transcription of 2 μg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Alameda, USA). Transcribed cDNA was used as a template for qRT-PCR. Using ACTB as a reference gene, qRT-PCR was performed in triplicate by the LightCycler FastStart DNA Master SYBR Green and LightCycler detection system (Roche Applied Science). The primers used for qRT-PCR were human ABCB4: 5′-GAGAGGACACAAACCAGACAGCA-3′ (forward); 5′-GTCTGCCCACTCTGCACCTT-3′ (reverse); human ACTB: 5′-CAGAAGGATTCCTATGTGG-3′ (forward); 5′-CATGATCTGGGTCATCTTC-3′ (reverse). The relative quantitative expression was calculated via the 2^−ΔΔCt method.