Genomic DNA from 5 males was extracted using the NucleoSpin Tissue XS kit (Macherey-Nagel, ref #740901). To amplify the Rsp satDNA region, we used two sets of primers: 5’-CCAGGCGAACAGAAGATACC-3’ and 5’-TTTTGACCGCTTAAAATGACA-3’; and 5’-AAGTTATGTCATTTTAAGCGGTCA-3’ and 5’-AACTTAGGCAATTTACTGTTTTTGC-3’. As a control, we amplified a fragment into nup62 gene (primers 5’-GGCACCTACTGCTGGTATCG-3’ and 5’-AATCCAAAGGCTGGTGGAG-3’). Quantitative PCR analysis was performed with 5ng of template gDNA in a 25μl reaction using the TB Green Premix Ex Taq II (Takara, ref #RR820L) and the CFX Connect (Biorad CFX Connect) system. For each set of primers, standard and melting curve analyses were performed to check for, respectively, PCR efficiency and specificity. qPCR analysis was done using technical duplicates on three biological replicates. The nup62 gene was used as internal control with a known copy number (two) so that genomic DNA levels were normalized for each sample to the levels of nup62. Based on [28 (link)], we considered that cn1bw1/SD-Mad flies carry 1000 repeats. The copy number in the Rsp satDNA is relative to cn1bw1/SD-Mad and was calculated using the comparative quantification ΔΔCT method [66 (link)].
Cfx connect
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The CFX Connect is a thermal cycler designed for real-time PCR (polymerase chain reaction) applications. It features a 96-well sample block and supports a variety of fluorescent detection chemistries. The CFX Connect provides precise temperature control and reliable performance for nucleic acid amplification and detection.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (105 mentions)
Trizol, by Thermo Fisher Scientific (52 mentions)
Iscript cdna synthesis kit, by Bio-Rad (40 mentions)
Rneasy mini kit, by Qiagen (37 mentions)
Itaq universal sybr green supermix, by Bio-Rad (33 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (26 mentions)
Iq sybr green supermix, by Bio-Rad (22 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (21 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (19 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (16 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (15 mentions)
Cfx manager software, by Bio-Rad (15 mentions)
Primescript rt reagent kit, by Takara Bio (15 mentions)
Superscript 3, by Thermo Fisher Scientific (15 mentions)
Sybr green, by Bio-Rad (14 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (14 mentions)
Nanodrop, by Thermo Fisher Scientific (12 mentions)
Rneasy kit, by Qiagen (12 mentions)
Sybr green supermix, by Bio-Rad (10 mentions)
Tb green premix ex taq 2, by Takara Bio (10 mentions)
733 protocols using cfx connect
1
Quantitative Analysis of Rsp Satellite DNA
2
Exosomal RNA Isolation and Quantification
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For the exosome-purified pellet, 35 μL of QIAzol was added and proceeded for total RNA isolation including both mRNA and miRNA using miRNeasy Micro Kit (Qiagen, #1071023, Germany) following the provided protocol. For mRNA analysis, weverse transcription was performed to synthesize cDNA using PrimeScript 1st strand cDNA synthesis kit (TAKARA, #6110A, JAPAN). Real-time qPCR for STC2 mRNA was performed with BioRad CFX Connect using SsoAdvance Universal Supermix (BioRad, #1725270, USA). For miRNA analysis, reverse transcription was performed with 10.0 ng RNA using miR-184-specific reverse transcription primer and quantified with miR-184 probe-based real-time qPCR using TaqMan MicroRNA Reverse Transcription Kit (Applied Bioscience, #4366596, US), miR-184-specific TaqMan MicroRNA Assay (Applied Bioscience, #4427975, US), and TaqMan Universal Master Mix II (Applied Bioscience, #4440040, US) following the manufacturer’s protocol. Real-time qPCR was performed with BioRad CFX Connect and analyzed with CFX Maestro software.
3
VCAM-1 Gene Expression Quantification
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Total RNA was extracted from the ECs by using TRIzol reagent (Invitrogen, Carlsbad, CA) and used as a template for cDNA synthesis. cDNA, obtained with High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA), was amplified by PCR on a CFX Connect (Bio-Rad) with Power SYBR Green PCR Master Mix (Applied Biosystems) and primers for VCAM-1 (100 nM, sense: 5′-ACGTCAGAACAACCGAATCC-3′; antisense: 5′-GTGGTGCTGTGACAATGACC-3′) and β-actin (sense: 5′-CTGGGTCATCTTTTCACGGT-3′; antisense: 5′-TGTTACCAACTGGGACGACA-3′) used as an internal control. The real-time PCR was conducted at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 10 sec, annealing/extension at 60 °C for 1 min. PCR conditions were optimised to achieve a single peak by melting curve analysis on CFX Connect (Bio-Rad). Raw data collected by use of CFX Manager were analysed and quantified by the relative standard curve method.
4
Quantification of ARG2 Expression in HL-60 Cells
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Total RNAs was extracted from indicated cells using TRIzol reagent (Qiagen). The concentration of cell-free total RNAs was quantified using NanoDrop 2000 UV-spectrophotometer (Thermo Scientific) and reverse transcribed into complementary DNA (cDNA) by using a Reverse Transcription Supermix kit (Bio-Rad, USA). The cDNA was then amplified with a pair of forward and reverse primers for the genes listed in Supplementary Data 1 created by using Microsoft Excel (version 2016). Results were normalized to the housekeeping gene β-Actin. The thermal cycling conditions composed of an initial denaturation step at 95 °C for 1 min followed by 35 cycles of PCR using the following profile: 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s using Bio-Rad CFX connect.
ARG2 duplex RT-qPCR was performed on HL-60 cells treated with or without nicotine in presence or absence of natural compound library (n = 124) using Taqman Gene Expression Cells-to-CT kit as per manufacturer’s recommendation on Bio-Rad CFX connect. Results were normalized to the housekeeping gene β-Actin labeled with VIC dye.
ARG2 duplex RT-qPCR was performed on HL-60 cells treated with or without nicotine in presence or absence of natural compound library (n = 124) using Taqman Gene Expression Cells-to-CT kit as per manufacturer’s recommendation on Bio-Rad CFX connect. Results were normalized to the housekeeping gene β-Actin labeled with VIC dye.
5
Quantitative RT-PCR Analysis of Gene Expression
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After transfection for 48 hours, total RNA was collected with an ultrapure RNA kit, and RNA was reversely transcribed into cDNA using a HiFiScript cDNA synthesis kit. cDNA was amplified using fluorogenic quantitative PCR (CFX Connect™; Bio-Rad, Hercules CA, USA). Primers (listed in Table 2 ) were added to 25 µL of PCR mix (9.5 µL of RNase free dH2O, 1 µL of cDNA, 1 µL of forward primer, 1 µL of reverse primer, 12.5 µL of 2× UltraSYBR mix) and reaction parameters were as follows: pre-denaturation for 10 minutes at 95°C, denaturation for 10 seconds at 95°C, annealing for 30 seconds at 55°C, elongation for 30 seconds at 72°C. Dissociation curves were analyzed as follows: 15 seconds at 95°C, 1 minute at 55°C, 15 seconds at 95°C, 15 seconds at 55°C, 15 seconds at 55°C, and measured stepwise from 92°C, every 0.5°C. Quantitative analysis was performed using fluorogenic quantitative RT-PCR (CFX Connect™; Bio-Rad). Glyceraldehyde-phosphate dehydrogenase served as an internal control.
6
Quantitative PCR for Gene Expression
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We extracted RNA from the cells or articular cartilages and reverse transcribed into cDNA in accordance with the instructions of regents or kits. Fluorogenic quantitative PCR (CFX Connect, Bio-Rad, Hercules, CA, USA) was performed to amplify cDNA. The PCR reaction system (20 µL) consisted of RNase-free distilled water (7 µL), cDNA/DNA (1 µL), forward primer (1 µL), reverse primer (1 µL), and 2× UltraSYBR Mixture (10 µL). Table 1 lists the sequences of forward and reverse primers that were synthetized by General Biosystems (Anhui, China). The PCR parameters consisted of pre-denaturation for 10 min at 95 ℃, denaturation for 10 s at 95 ℃, annealing for 30 s at 58 ℃, elongation for 30 s at 72 ℃, and 40 cycles. Parameters for dissociation curve analysis consisted of 15 s at 95 ℃, 1 min at 58 ℃, 15 s at 95 ℃, 15 s at 8 ℃, and 15 s at 58 ℃. They were measured stepwise from 95 ℃ every 0.5 ℃. The products of PCR were evaluated on an RT-PCR system (CFX Connect, Bio-Rad, USA). We used GAPDH and U6 as internal controls. The relative expression of genes was calculated via the 2−ΔΔCt method (28 (link)).
7
Quantitative Real-Time PCR Analysis
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Samples were collected from seedlings and frozen until analysis. RNA was isolated from frozen tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA samples were first treated with DNase I and then an MMLV First-Strand Synthesis Kit (Gene Direx, Grand Island, NY, USA) was used to synthesize the first strand of cDNA [reverse transcription (RT)]. Using membrane-anchored ubiquitin-fold (MUB) proteins as the internal control, this study performed quantitative real-time (qRT) PCR with specific primer pairs (Supplementary Table 1 ). Reactions were conducted using a Bio-Rad real-time PCR detection system (CFX ConnectTM, Bio-Rad, USA), and relative expression was analyzed using Bio-Rad CFX Manager 3.1 software (CFX ConnectTM, Bio-Rad). The ubiquitin gene was used as an internal control to normalize the cDNA levels. The reaction conditions were 94 °C for 5 min and 45 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Independent tests were performed at least three times, and the results were averaged. The sequences of primers used for qRT-PCR are presented in the supplementary information (Additional file 1 ).
8
Quantification of HERV-K and HERV-W env in Brain Tissue
9
Quantitative Analysis of Lipoprotein Receptor mRNA
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Hepatocyte lysates were collected, after transfection and apo(a)/Lp(a) treatment, using RLT Plus buffer (Qiagen) supplemented with 1% (v/v) 2-mercaptoethanol. Total RNA was isolated from the lysates using the RNeasy Plus kit (Qiagen) as per the manufacturer’s protocol. LRP-8, LRP-1 or VLDLR mRNA expression was assessed by qRT-PCR utilizing primer pairs A/B, C/D, E/F, respectively (Table 1 ) and normalized to GAPDH using primer pair G/H (Table 1 ). All DNA primers utilized in this study were synthesized by Integrated DNA Technologies, Inc. Reactions were assembled in triplicate using the iTaq Universal SYBR Green One-Step kit (Bio-Rad) according to the manufacturer’s protocol, and performed on a Bio-Rad CFX Connect. Relative quantification of the respective mRNA was determined by normalization of the threshold cycle for each sample with that of GAPDH (primer set G/H; Table 1 ) from the same reaction well using Bio-Rad CFX Manager software version 3.1 (Bio-Rad). All mRNA expression data are reported as a fold-increase compared with the control sample from cells that were transfected with the pCMV6 parental plasmid.
10
Gene Expression Analysis of Adipose Tissue
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