Verity thermal cycler

Expression profiling of 188 miRNAs identified as the most consistent and reliable miRNAs in human plasma^{14 (link)} was performed using TaqMan-Low-Density-Array (TLDA) human custom arrays as instructed by the manufacturer (Applied Biosystem, CA, USA) with Applied Biosystems QuantStudio 7. Briefly, a fixed volume of 3 μL of miRNA solution was used for reverse transcription using a TaqMan MicroRNA Reverse Transcription Kit and the TaqMan Custom RT pool (Applied Biosystems, CA, USA), which were customized for TLDAs. The reaction was performed under the following conditions: incubation for 30 min at 16 °C and 30 min at 42 °C, inactivation for 5 min at 85 °C and immediate cooling to 4 °C. The cDNA was stored at − 20 °C. For our custom selection, 3.125 μL of the reverse transcription reaction was used for preamplification using TaqMan PreAmp Master Mix (2X) and TaqMan PreAmp pool (Applied Biosystems, CA, USA). The reaction conditions were as follows: 10 min at 95 °C, 2 min at 55 °C, 2 min at 72 °C, 12 cycles of 15 s at 95 °C and 4 min at 60 °C, inactivation for 10 min at 99.9 °C and cooling to 4 °C. The preamplification reaction was then diluted with water (1:3), and 4.5 μL was used for TLDA. Reverse transcription and preamplification reactions were carried out with an Applied Biosystems Verity Thermal Cycler. Data were normalized by the mean-centre normalization method.

A multiplex reverse transcription-PCR (RT-PCR) was performed using the OneStep RT-PCR kit (Qiagen) on a Verity Thermal Cycler (Applied Biosystems) with the following GAPDH forward (FW) and reverse (R) primers in combination; hGAPDH_FW: 5′-CGA CAG TCA GCC GCA TCT T-3′ (h for human), hGAPDH_R1: 5′-CCC CAT GGT GTC TGA GCG-3′, product size with FW-primer: 62 bp, hGAPDH_R2: 5′-AAG CAG CCC TGG TGA CCA G-3′, product size with FW-primer: 123 bp, hGAPDH_R3: 5′-GCC ATG GAA TTT GCC ATG GG-3′, product size with FW-primer: 230 bp, hGAPDH_R4: 5′-CCA GCA TCG CCC CAC TTG A-3′, product size with FW-primer: 328 bp. This cocktail of primer pairs for amplification of GAPDH RT-PCR products of varying lengths was used to evaluate the quality of RNA extracted from sections of FFPE blocks. SYBR safe DNA gel stain (Thermo Fisher Scientific, Hvidovre, Denmark) and Gel loading dye (BioNordika, Herlev, Denmark) was used to visualize RT-PCR products using a Fuji LAS-3000 Imaging System (Fujifilm Europe GmBH, Düsseldorf, Germany) with IMAGE READER LAS-3000 software version 2.2 (Science Imaging Scandinavia AB, Saltsjö-Boo, Sweden).

RNeasy Plus Mini kit (QIAGEN, #74134) was used for purifying total RNA from pre-cultured (t = 0) and differentiated cells (t = 10 and 21 days). RNA concentration was determined using a Nanodrop 2000 UV-Vis spectrophotometer (Thermo). SuperScript VILO Mastermix (Invitrogen, #11755250) was used for obtaining cDNA from 350 ng RNA in a total reaction volume of 20 µL. A Verity Thermal Cycler (Applied Biosystems) was programmed as follows: 10 minutes at 25°C, 60 minutes at 42°C and 5 minutes at 85°C. qPCR reactions were performed in a Applied Biosystems 7500 Fast Real Time PCR System using TaqMan Gene Expression Mastermix (Applied Biosystems, #4369510), according to manufacturer's instructions. Oligonucleotides and probes for qPCR were purchased from Applied Biosystems (TaqMan gene expression assay, #4331182): HPRT1 (Hs02800695_m1), RPL13A (Hs03043885_g1), SOX2 (Hs01053049_s1), POU5F1 (Hs00999634_gH), TERT (Hs00972656_m1), PPARG (Hs01115513_m1), ADIPOQ (Hs00605917_m1), CEPBA (Hs00269972_s1), BMP2 (Hs00154192_m1), SPARC (Hs00234160_m1), RUNX2 (Hs00231692_m1), SOX9 (Hs00165814_m1), ACAN (Hs00153936_m1) and COL2A1 (Hs00264051_m1).

RNA was extracted from cell culture pellets and sections of paraffin-embedded tissue samples using the RNeasy Mini Kit and the RNeasy FFPE Kit (both Qiagen, Copenhagen, Denmark), respectively. An additional on-column DNase (Qiagen) digestion step was performed to remove genomic DNA. mRNA was converted into cDNA using the High-Capacity cDNA Reverse Transcription Kit on a Verity Thermal Cycler (both Applied Biosystems, Naerum, Denmark). gDNA background was evaluated in no reverse transcriptase control reactions.

To develop a fine map around candidate regions for the resistance genes and investigate the relationship with previously anthracnose resistance genes mapped in similar genetic positions, an expanded set of molecular markers was also analyzed: i) markers previously linked to anthracnose resistance genes located on Co-1 and Co-3 clusters [for more details see S1 Table]; ii) InDel markers (Insertion-deletion polymorphisms), based on their physical positions on the bean genome [40 (link)]; iii) microsatellites specifically designed from the P.vulgaris genomic sequence using the software BatchPrimer3 and Oligo Anlyzer [41 (link)].
PCR amplification was performed in a Verity Thermal Cycler (Applied Biosystems, Life Technologies, CA, USA) in a final volume of 20 μL solution containing 25 ng of genomic DNA, 100 mM Tris–HCl, 100 mM KCl (pH 8.3), 4 mM MgCl2, 0.2 mM each dNTP (Bioline, London, UK), 0.2 μM each primer, and 1.25 U of Biotaq DNA polymerase (Bioline). Amplification products were resolved on polyacrylamide (8%) or agarose gels (1.5%) in 1x TBE buffer (89 mM TRIS, 89 mM boric acid, 2 mM EDTA), stained with RedSafe and visualized under UV light.

RNA was isolated from adult mouse tissues and embryos using TRIzol Reagent (Thermo-Fisher) following the manufacturers protocol. Five micrograms of glycogen was added to the aqueous phase prior to addition of isopropanol to facilitate RNA precipitation. RNA concentration was determined using a Nanodrop spectrophotometer. cDNA was prepared with a Versco cDNA synthesis kit, using 3 μg of RNA as template plus 200 ng of random hexamer and 200 ng oligo dT as primers, following the manufacturer’s instructions. All PCR reactions were optimized for each primer set by gradient PCR, using an Applied Biosystems Verity thermal cycler. PCR reactions contained 1 μl of cDNA, primer sets (S2 Table) and GoTaq Green master mix (Promega) following the manufactures protocol. PCR products were size fractionated on 1% agarose gels and subsequently the bands were isolated, DNA was purified from the gel using Micro Bio-Spin columns (Bio-Rad) and subjected to sequencing. All Sanger DNA sequencing was performed by the Center for Applied Genomics (CAG; caglab.org) at the Children's Hospital of Philadelphia.