Total RNA from WT and MCT4-KO MEFs exposed to CTRL- or 5% CSE-media was isolated using TRIzol® Reagent (15596026; Invitrogen) following manufacturer’s instructions. Total RNA concentration and purity was determined using NanoDrop® ND-1000 spectrophotometer (Thermo Scientific). Reverse transcription polymerase chain reaction (RT-PCR) was performed using a High-Capacity cDNA Reverse Transcription Kit (4368814; Applied Biosystems) and carried out in a Bio-Rad PTC-100® thermal cycler. cDNA obtained was used to perform real-time PCR target amplification. Quantitative Polymerase Chain Reaction (qPCR) master mixes were prepared using TaqMan Universal PCR Master Mix (4304437; Applied Biosystems) according to manufacturer’s recommendations and loaded into MicroAmp® Optical 384-Well reaction plates (4483319; Applied Biosystems). Inventoried TaqMan® Gene expression assays CCL2 (Mm00441242_m1), β-actin (Mm00607939_s1) and GAPDH (Mm00607939_s1) were used. Each qPCR reaction was performed in triplicates. Plates were analyzed in a QuantStudio 12K Flex Real-Time PCR System (Thermo Scientific). mRNA expression levels were calculated using the relative comparison CT (2-ΔΔCT) method after normalization with GAPDH as endogenous control.
Microamp optical 384 well reaction plate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MicroAmp Optical 384-well reaction plate is a laboratory equipment designed for use in real-time PCR and other high-throughput applications. The plate features 384 individual wells to accommodate multiple samples simultaneously. The optical properties of the plate are optimized for fluorescence detection during PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Viia 7 real time pcr system, by Thermo Fisher Scientific (8 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Faststart universal sybr green master rox, by Roche (3 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (3 mentions)
Quantstudio 7, by Thermo Fisher Scientific (3 mentions)
Sds v2, by Thermo Fisher Scientific (3 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Sybr select master mix, by Thermo Fisher Scientific (2 mentions)
Quantstudio 5, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Rnase free water, by Qiagen (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Ptc 100 thermal cycler, by Bio-Rad (1 mentions)
34 protocols using microamp optical 384 well reaction plate
1
Quantifying mRNA Expression in MEFs
2
Optimization of qPCR Methods for Measuring Mitochondrial DNA Content
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ρ0 cells have mtDNA-CN close to zero (0.1 is typically reported in HeLa ρ0) (10 ). We aimed to have a similarly low number in our BET-1A and BEAS-2B ρ0 cells.
The qPCR reaction was performed in triplicate for each sample at a volume of 20 μl per well. Each qPCR reaction contained 2 μl diluted sample DNA (varying concentrations of DNA tested as described below), 10 μl Power-SYBR Green Polymerase Chain Reaction (PCR) Master Mix (Applied Biosystems), 6 μl PCR water, and 2 μl primer mix containing 10 nM forward and reverse primers. qPCR reactions were performed on Applied Biosystems QuantStudio5 using MicroAmp Optical 384-Well Reaction Plates. The real-time PCR conditions were the following: initial denaturation 2 minutes at 50°C ramp to 95°C for 10 minutes followed by 40 amplification cycles of (95°C for 15 seconds denaturation ramp down to 60°C for 60 secs for annealing and extension) followed by a final 95°C for 15 second hold. The cycle threshold values (Ct values) were determined automatically via QuantStudio Design & Analysis software.
To optimize the qPCR methods, we tested several primer sets detecting both mtDNA and nDNA. Primer sets tested are reported inTable 1 . For this purpose, master mix was prepared by mixing DNA, SYBR-Green and PCR water, and then added to the PCR plates which contain the 2 μL of primer sets.
The qPCR reaction was performed in triplicate for each sample at a volume of 20 μl per well. Each qPCR reaction contained 2 μl diluted sample DNA (varying concentrations of DNA tested as described below), 10 μl Power-SYBR Green Polymerase Chain Reaction (PCR) Master Mix (Applied Biosystems), 6 μl PCR water, and 2 μl primer mix containing 10 nM forward and reverse primers. qPCR reactions were performed on Applied Biosystems QuantStudio5 using MicroAmp Optical 384-Well Reaction Plates. The real-time PCR conditions were the following: initial denaturation 2 minutes at 50°C ramp to 95°C for 10 minutes followed by 40 amplification cycles of (95°C for 15 seconds denaturation ramp down to 60°C for 60 secs for annealing and extension) followed by a final 95°C for 15 second hold. The cycle threshold values (Ct values) were determined automatically via QuantStudio Design & Analysis software.
To optimize the qPCR methods, we tested several primer sets detecting both mtDNA and nDNA. Primer sets tested are reported in
3
RNA Extraction and Quantitative PCR
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Total RNA was extracted using QIAshredder spin columns and an RNeasy Plus Micro kit or Mini kit (QIAGEN, Manchester, UK) as per manufacturer’s instructions. For quantitative PCR, purified RNA was reverse-transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit following manufacturer’s instructions (Thermo Fisher). qPCR reactions were performed in triplicate from cDNA with a concentration between 1 and 25ng, and expression of the housekeeping gene ACTB was used for normalization. The qPCR assays were executed using 2x SYBR Green Mastermix (Thermo Fisher) in MicroAmp® optical 384-well reaction plates (Applied Biosystems) and analyzed using an Applied Biosystems 7900HT Sequence Detection System. Primers used are shown in Table S4 . ΔCt values relative to ACTB were assessed using SDS software v2.1 (Applied Biosystems).
4
Quantitative PCR Protocol for Gene Expression
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Total RNA was extracted using the RNeasy Mini Kit with DNAse digestion (Qiagen,
West Sussex, UK). cDNA was prepared from ∼1 µg of RNA using the High Capacity
Reverse Transcriptase kit (Applied Biosystems, California, USA), according to
the manufacturer's instructions. All qPCR reactions were prepared in MicroAmp®
optical 384-well reaction plates (Applied Biosystems) using 50 ng/µl cDNA with
SYBR®Green Jumpstart™ Taq Readymix™ (Sigma-Aldrich), ROX reference dye
(Invitrogen) and sense and anti-sense primers (all 200 nM). Primers for human:
BMPR2, ACTB; mouse: Bmpr2, Actb, B2m and
Hprt were all designed using Primer3 (http://primer3.sourceforge.net/ ). Reactions were amplified on a
QuantStudio 6Flex Real-Time PCR system (Applied Biosystems). In human BOECs,
target gene expression was normalised to ACTB and the
difference in the amount of product produced was expressed as a fold change.
Relative expression of each target gene was identified using the comparative
2-(ΔΔCt) method. In mouse lung, target gene expression was normalised to
Actb, B2m and Hprt and the difference
expression represented as relative expression.
West Sussex, UK). cDNA was prepared from ∼1 µg of RNA using the High Capacity
Reverse Transcriptase kit (Applied Biosystems, California, USA), according to
the manufacturer's instructions. All qPCR reactions were prepared in MicroAmp®
optical 384-well reaction plates (Applied Biosystems) using 50 ng/µl cDNA with
SYBR®Green Jumpstart™ Taq Readymix™ (Sigma-Aldrich), ROX reference dye
(Invitrogen) and sense and anti-sense primers (all 200 nM). Primers for human:
BMPR2, ACTB; mouse: Bmpr2, Actb, B2m and
Hprt were all designed using Primer3 (
QuantStudio 6Flex Real-Time PCR system (Applied Biosystems). In human BOECs,
target gene expression was normalised to ACTB and the
difference in the amount of product produced was expressed as a fold change.
Relative expression of each target gene was identified using the comparative
2-(ΔΔCt) method. In mouse lung, target gene expression was normalised to
Actb, B2m and Hprt and the difference
expression represented as relative expression.
5
Quantitative PCR for Gene Expression
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Gene primer sequences were designed with Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and synthesized by Sigma-Aldrich (Table S2 ). For conventional qPCR, 1.5 mL of cDNA were mixed with specific primers and SYBR Select master mix (4472908, Invitrogen) constituting a final volume of 6.5 mL, in MicroAmp Optical 384-Well Reaction Plates (4309849, Applied Biosystems). Each reaction was performed in triplicate using the ViiA 7 Real-Time PCR system (Applied Biosystems). qPCR conditions involved an initial denaturation step (90 s at 95°C), followed by 40 cycles of annealing (15 s at 95°C and 1 min at 59°C), and a final extension phase (15 s at 95°C, 1 min at 60°C and 15 s at 95°C). Ct values were extrapolated from the melt curve and gene expression levels were normalized with RPLP0 or Gapdh housekeeping expression by implementing the 2ΔΔCt formula.
6
Taqman MicroRNA Expression Analysis
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For each sample, 5 ng of total RNA was used in a 15 μl reverse transcription reaction using Taqman miRNA Reverse Transcription Kit (Life Technologies, Grand Island, NY, USA) and miRNA-specific primers according to the manufacturer’s instructions. The reverse transcription product (1.33 μl) was used in a 20 μl Taqman MicroRNA Taqman Assay reaction with predesigned miRNA-specific probes, according to the manufacturer’s instructions. Taqman PCR was conducted in MicroAmp Optical 384-well reaction plates (Applied Biosystems, Waltham, MA, USA) on an ABI 7900HT real-time PCR detection system. The expression level of each sample for each miRNA was standardized to U6 small nuclear RNA (snRNA), to control for differences in RNA loading, quality, and complementary DNA (cDNA) synthesis using the ΔΔCt method. For graphing purposes, the log2 relative expression levels are shown, such that the expression level of the mean expression for Taqman PCR (n = 5) and microarray data (n = 4 or 5) is equal to zero. A Pearson correlation coefficient (R) was calculated for each gene comparing Taqman and microarray relative expression with age.
7
Quantitative Real-Time PCR Analysis
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Quantitative real-time PCR (qRT-PCR) analysis was performed using ABI MicroAmp Optical 384-well reaction plates on an ABI 7900 real-time PCR instrument (Applied Biosystems) and Taq Pro Universal SYBR qPCR Master Mix (Q712, Vazyme Biotechnology Co., Ltd., Nanjing, China). The qRT-PCR protocol included 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 60 °C for 30 s after a pre-denaturation stage at 95 °C for 30 s. Gapdh, β-Actin, and Rpl41 were used as the housekeeping gene to normalize all the results, which were presented as 2−ΔΔCt. The primers used were designed on the online website Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/ , accessed on 15 February 2023) and are listed in Supplementary Table S1 .
8
Quantitative RT-PCR Analysis of Aquaporin, HIF-1α, and PGK1
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Tissue slices of 50µm were shredded and lysed in Buffer RLT Plus (QIAGEN). RNA isolation was subsequently performed using the RNeasy Plus Mini Kit (QIAGEN, Cat. No. 74134) according to the manual. RNA was eluted in 30 µL or 14 µL nuclease-free water, respectively. RNA concentration was determined using a Colibri Microvolume Spectrometer (BioConcept AG). cDNA synthesis was performed using the GoScript™ Reverse Transcription System (Promega, Cat. No. A5000) from up to 200 ng RNA per reaction. A Biometra T-Personal Thermal Cycler was used. Quantitative PCR was performed using the FastStart Universal SYBR Green Master (Rox) (Roche, Cat. No. 4913850001). AQP1, HIF-1α, and PGK1 specific primers (Microsynth, Balgach, Switzerland) were used for the amplification of cDNA. Per reaction, 5 µL Master Mix was mixed with primers forward and reversed (0.5 µM), 1 µL of cDNA template or water in NTC, and water to a final volume of 10 µL. PCR reactions were performed in triplicates in MicroAmp™ Optical 384-Well Reaction Plates (Applied Biosystems, Cat. No. 4309849) in a ViiA 7 Real-Time PCR System (Applied Biosystems) using the associated software. Data were analyzed using the 2−ΔCT method × 1000.
9
Screening of Diverse Small Molecule Library
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A library of 107 008 diverse small molecule compounds (AstraZeneca) in DMSO was arrayed, in pools of four compounds, in MicroAmp Optical 384-well reaction plates (Applied Biosystems, ThermoFisher Scientific). The compounds were selected as a representative subset of the entire corporate 2.2 million compound screening library to provide maximal chemical diversity, while retaining good physicochemical properties (see https://openinnovation.astrazeneca.com/target-innovation.html # ).
10
Quantitative Real-Time PCR Analysis
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Mus musculus gene primer sequences were designed with the Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and synthesized by Sigma-Aldrich. DNA (1.5 μl) was mixed with specific primers (Table S1 ) and SYBR Select Master Mix (4472908, Invitrogen) constituting a final volume of 6.5 μl, in MicroAmp Optical 384-Well Reaction Plates (4309849, Applied Biosystems). Each reaction was performed in duplicate using the ViiA 7 Real-Time PCR System (Applied Biosystems). qPCR conditions involved an initial denaturation step (90 s at 95ºC), followed by 40 cycles of annealing (15 s at 95ºC and 1 min at 59ºC), and a final extension phase (15 s at 95ºC, 1 min at 60ºC and 15 s at 95ºC). Ct values were extrapolated from the melting curve and gene expression levels were normalized with GAPDH or HPRT, or 18S ribosomal RNA housekeeping expression by implementing the formula.
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