Rotor gene 6000 thermocycler

Cells were transfected with miRNA precursors (30 nM) or siRNAs (5 nM) for 24 h and RNA isolated using Qiazol. For cDNA synthesis, 0.5 μg of total RNA was reverse transcribed using QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR was performed with a Rotor-Gene 6000 thermocycler (Qiagen) or ViiA7 Real-Time PCR system (Life Technologies), using SensiMixPlus SYBR (Bioline). Primer sequences are listed in Supplementary Table S8. Validated primer sequences from PrimerBank were used where available [65 (link)]. Relative expression for each sample was calculated, taking into account PCR efficiency [66 (link)] with GAPDH or HPRT1 used as reference genes. A NF-κB pathway PCR array (RT² Profiler PCR Array, SABiosciences, Qiagen) was performed with WM266-4 cells transfected with miR-NC or miR-7-5p for 72 h, according to manufacturer's instructions and analyzed with the PCR Array Data Analysis Software provided from the company.

Quantitative PCR (qPCR) was used to examine the temporal expression of Chl1 within the developing VM. In brief, the VM was isolated from E10.5, E12.5, E14.5 and E18.5 embryos (4 litters per age) as previously described^{7 (link), 18 (link)}. The VM was additionally isolated from tyrosine hydroxylase green fluorescent protein (TH-GFP+) embryos at E12.5^{19 (link)}, to establish whether Chl1 was expressed on DA neurons (FACS isolated THGFP+ cells) or non-dopaminergic neurons (THGFP− cells) within the VM (n = 4 embryos per FACS preparation, repeated on 4 litters). For all samples RNA was isolated from using RNeasy Micro kit (Qiagen) and reverse transcribed using a SuperScript® VILO™ cDNA Synthesis Kit. qPCR performed using SYPR GreenER qPCR SuperMix Universal (Invitrogen) on a Rotor-Gene 6000 thermocycler (QIAGEN) and analyzed using the comparative ΔΔCT method^{20 (link)}. Oligonucleotide sequences were as follows:
Hprt forward, 5′-CTTTGCTGACCTGCTGGATT-3′
Hprt reverse, 5′-TATGTCCCCCGTTGACTGAT-3′
Chl1 forward, 5′-TGGAATTGCCATTATGTGGA-3′
Chl1 reverse, 5′-CACCTGCACGTATGACTGCT-3′

Quantitative PCR (qPCR) of the 23S-rRNA gene was performed as described in [27 (link)] to quantify phototrophs. qPCR was done in reactions containing 1 µL of sample gDNA, 1 µL of each forward/reverse primer (1.25 µM each), 5 µL of SYBR Green ssoAdvanced PCR Mix (Qiagen, Venlo, Netherlands), and DNAse-free water up to 10 µL (Qiagen, Venlo, Netherlands) on a Rotor-Gene 6000 thermocycler (QIAGEN, Venlo, Netherlands). Based on a search of the MicroGreen 23S rRNA gene database, the 23S rRNA gene primers used were determined as universal to phytoplankton, matching 1942 of 2326 total database sequences [34 (link)]. Standards were constructed from a Cryptomonas 23S rRNA gene sequence, which was PCR-amplified as described above from BML water samples and then cloned into a PJET 3.0 plasmid (ThermoFisher, Waltham, MA, USA) based on the CloneJET PCR Cloning Kit protocol (Thermo Scientific). The plasmids were PCR-amplified using the 23S rRNA gene primers, then amplicons were quantified with a Qubit HS kit (Invitrogen, Carlsbad, CA, USA) and serially diluted over 7 orders of magnitude. The detection limit was ~ 100 gene copies mL⁻¹ and median amplification efficiency was 89%. Examination of qPCR melt curves suggested primer specificity.

Bacterial DNA was amplified by quantitative PCR (qPCR) with the bacterial 16S rRNA gene primers F_Bact 1369 (5′-CGG TGA ATA CGT TCC CGG-3′) and R_Prok 1492 (5′-TAC GGC TAC CTT GTT ACG ACT T-3′) (Suzuki, Taylor & DeLong, 2000 (link)), using a Rotor-gene 6,000 thermocycler (Qiagen, Hilden, Germany). Samples were measured in duplicate using 10 µL reactions consisting of 1 µL DNA template, 0.25 µL forward primer (10 pmol/µL), 0.25 µL reverse primer (10 pmol/µL), 3.5 µL nuclease-free water, and 5 µL of KAPA SYBR^® FAST Universal 2X qPCR Master Mix. Cycling conditions consisted of 95 °C for 3 min, followed by 40 cycles of (95 °C for 20 s, 60 °C for 30 s, and 72 °C for 30 s). Calculated concentrations (ng/µL) were normalized to extracted DNA concentrations. The amount of DNA detected was expressed as equivalent number of Escherichia coli genomes per ng of total DNA to provide an estimate of the numbers of bacteria present.

A Taqman RT-qPCR assay was used to quantify the steady-state mRNA levels of EWSR1–FLI1, NR0B1, and TBP (used as a reference housekeeping gene) as described previously [37 (link)]. The CD44 mRNA steady-state levels were quantified using the SYBR-Green qPCR kit (Quantimix Easy Kit, #10606-4153, Biotools, Madrid, Spain). The PCR reactions were run on a Rotor Gene 6000 thermocycler (Qiagen, Hilden, Germany). The cycle threshold (Ct) for each gene and TBP was calculated using the RotorGene Software (v2.3.1). The relative expression for each gene was calculated as 2^−∆Ct, where ∆Ct = Ct_gene − Ct_TBP. Sequences of primers and TaqMan probes are shown in Supplementary Materials Table S3.

A single-tube nested real-time PCR assay^{38 (link)} which is based on the CocciEnv real-time PCR target^{39 (link)} was run in duplicate to analyze DNA samples for presence of Coccidioides DNA. At lab A, each 10 µL reaction mixture contained 2 µL DNA template, 5 µL 2× SSOAdvanced Universal Probes Supermix (Bio-rad), 240 nM each of primers and probe, and 2.5 µL nuclease-free water. Thermocycling conditions consisted of an initial denaturation for 10 min at 95 °C, followed by 11 outer amplification cycles at 95 °C 10 s, 65 °C 30 s, 72 °C 15 s, and 45 inner amplification cycles at 95 °C 10 s, 52 °C 30 s, 72 °C 15 s on a Biorad CFX Connect. At lab B, the assay was run in 20 µL reactions containing TaqMan Universal Polymerase Chain Reaction (PCR) Master Mix (Applied Biosystems, Grand Island, NY, USA), 240 nM each of primers and probe, BSA (2 ng/μl; BSA) and 2 μl DNA template. Thermocycling conditions consisted of an initial denaturation for 10 min at 95 °C, followed by 25 outer amplification cycles at 95 °C 10 s, 65 °C 30 s, 72 °C 15 s, and 45 inner amplification cycles at 95 °C 10 s, 52 °C 30 s, 72 °C 15 s on a Rotor-Gene 6000 thermocycler (Qiagen; Valencia, CA, USA). Samples were considered positive if they displayed a Ct less than 45 and displayed logarithmic amplification plots on at least one of the duplicate real-time PCR reactions.