Cardiac loops isolated from experimental and control (CFDA) embryos (Supplementary Figure S2 ) were subjected to qRT-PCR analysis following MIQE guidelines [72 (link),73 (link),74 (link)]. RNA was extracted and purified using the ReliaPrep RNA Cell Miniprep System Kit (Promega) according to the manufacturer’s instructions. For mRNA expression measurements, 1 μg of total RNA was used for retro-transcription with a Maxima First Strand cDNA Synthesis Kit for qRT-PCR (Thermo Scientific). Real-time PCR experiments were performed with 2 μL cDNA, Go Taq qPCR Master Mix (Promega) and corresponding primer sets (Supplementary Table S1 ). For microRNA expression analyses, 20 ng of total RNA was used for retro-transcription with Universal cDNA Synthesis Kit II (Exiqon) and the resulting cDNA was diluted 1/80. Real-time PCR experiments were performed with 1 μL of diluted cDNA, Go Taq qPCR Master Mix (Promega) as well. All qPCRs were performed using a CFX384TM thermocycler (Bio-Rad) following the manufacture’s recommendations. The relative expression of each gene was calculated using Gusb and Gadph as internal controls for mRNA expression analyses and 5S and 6U for microRNA expression analyses, respectively [75 (link)]. Each PCR reaction was carried out in triplicate and repeated in at least three distinct biological samples to obtain representative means.
Maxima first strand cdna synthesis kit for qrt pcr
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Maxima First Strand cDNA Synthesis Kit for qRT-PCR is a reagent kit designed for the reverse transcription of RNA into first-strand cDNA. It includes all the necessary components for efficiently converting RNA into cDNA, which can then be used as a template for quantitative real-time PCR (qRT-PCR) applications.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Dsdnase, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Reliaprep rna cell miniprep system kit, by Promega (1 mentions)
Cfx384 thermocycler, by Bio-Rad (1 mentions)
Universal cdna synthesis kit 2, by Qiagen (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Total rna mini kit, by Geneaid (1 mentions)
Maxima sybr green fluorescein qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Brilliant 3 ultra fast sybr qpcr mm, by Agilent Technologies (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mic real time pcr cycler, by Bio Molecular Systems (1 mentions)
Rneasy mini kit column, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
19 protocols using maxima first strand cdna synthesis kit for qrt pcr
1
Cardiac Loops qRT-PCR Analysis
2
RNA Isolation and qRT-PCR Analysis
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Cells treated with sgRNAs of interest were washed with PBS, pelleted and flash frozen. Total RNA was isolated from frozen cells using the RNeasy Mini kit (74104, Qiagen). Cells were resuspended as per manufacturer’s instructions for RNAse-rich cell lines using 2-MercaptoEthanol (Sigma-Aldrich, M6250-250 mL) and on-column DNAse digestion was performed to further purify the product (Qiagen, 79254). Isolated RNA was converted to cDNA using the Maxima First Strand cDNA Synthesis Kit for qRT-PCR as per manufacturer’s instructions (Thermo Fisher, K1641). Reactions were performed with the Dynamo ColorFlash SYBR Green qPCR kit (Thermo Fisher, F416L) per manufacturer’s instructions on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher, 4485701). The relative expression ratios were calculated relative to the housekeeping gene GAPDH using the 2−ΔΔCt method. Experiments were performed in technical triplicate with means and standard deviations plotted. Primers used are included in table S10 .
3
RNA Extraction and cDNA Synthesis
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Total RNA was isolated from skin samples or cell pellets using Total RNA Mini Kit (Geneaid Biotech, Cat. No. RT050, New Taipei City, Taiwan). The quality and quantity of the isolated RNA was determined with a NanoDrop ND-1000 spectrophotometer (Baylor College of Medicine, Houston, TX, United States). Thereafter, 500 ng total RNA was reverse-transcribed using Maxima First Strand cDNA Synthesis Kit for qRT-PCR (Thermo Fisher Scientific, Cat No. K1642) according to the manufacturer’s instructions.
4
Gene Expression Analysis of Pluripotency and EMT Markers
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Total RNA was extracted using GeneJET RNA Purification Kit (Thermo Fisher Scientific™, Waltham, MA, USA) following manufacturer’s instruction. 1 μg of RNA was reverse transcribed into cDNA using Maxima First Strand cDNA Synthesis Kit for qRT-PCR (Thermo Fisher Scientific™). qRT-PCR was carried out following the Maxima SYBR Green/Fluorescein qPCR Master Mix (Thermo Scientific™) protocol. The PCR setting were as follows: 5 min at 95 °C; 40 cycles of amplification at 95 °C for 15 s; 58 °C for 20 s; and 72 °C for 30 s. The primers amplifying SOX2, NANOG, OCT4, TWIST1, ZEB1, and SNAI1 are listed in Supplemental Table S1 . Gene expression levels were normalized by β-actin and quantified using the 2−ΔΔCt method.
5
Folic Acid Exposure Quantification
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The cells used for the folic acid experiments were grown in RPMI 1640 medium (Fisher, #27016021) supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin/streptomycin. The rest of the experiments were carried out using DMEM (Thermo Fisher, #15017CV) supplemented with 10% FBS, 2 mM L-glutamine, and 100 IU/mL penicillin/streptomycin. All the cells were maintained in a humidified incubator at 37°C with 5% CO2. Total mRNA was extracted from HeLa, MCF-7, and A549 using an RNeasy mini kit (QIAGEN) following the manufacturer’s instructions. mRNA was converted to cDNA using a Maxima First Strand cDNA Synthesis Kit for qRT-PCR (Thermo Fisher, #K1671).
qPCR was performed in the CFSX384 instrument (Bio-Rad) using Brilliant III Ultra-Fast SYBR QPCR MM (Agilent, #600882).
Primers: forward (hFOLR1) 5′-aca agg att gca tgg gcc ag-3′; reverse (hFOLR1) 5′-agg tgc cat ctc tcc aca gtg-3′; forward (hACTB) 5′-gtc acc aac tgg gac gac at-3′; reverse (hACTB) 5′-gta cat ggc tgg ggt gtt ga-3′.
qPCR was performed in the CFSX384 instrument (Bio-Rad) using Brilliant III Ultra-Fast SYBR QPCR MM (Agilent, #600882).
Primers: forward (hFOLR1) 5′-aca agg att gca tgg gcc ag-3′; reverse (hFOLR1) 5′-agg tgc cat ctc tcc aca gtg-3′; forward (hACTB) 5′-gtc acc aac tgg gac gac at-3′; reverse (hACTB) 5′-gta cat ggc tgg ggt gtt ga-3′.
6
Evaluating Gene Expression of MSCs on Ti Surfaces
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MSCs were seeded at passage 3 on Ti surfaces at 30,000 cells per sample for 3 and 7 days in order to evaluate the gene expression. After each time point, the cells were unattached and pellets were collected. Then, the cells were lysed and total RNA was extracted and purified using the RNeasy Mini Kit columns (Qiagen, Hilden, Germany) as described in the manufacturer’s instructions. Then, cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for qRT–PCR, with dsDNase (Thermo Fisher Scientific, #K1671). RT–PCR was carried out on a Mic real-time PCR cycler (Bio Molecular Systems, Australia), and gene expression was assessed by using the QuantiNova Fast SYBR™ Green PCR Master Mix (Qiagen). β-Actin was used as a housekeeping gene, and the relative gene expression levels were evaluated using the 2ΔΔ−Ct method. Primer sequences used are shown in Table 3 .
7
Transcriptomic analysis of salt stress response in Arabidopsis and Avicennia
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Total RNA was isolated from roots of control and treated (500 mM NaCl for 24 h) greenhouse-grown A. officinalis using Qiagen RNeasy kit (QIAGEN) and DNase treated (RNase-free DNase set, QIAGEN) according to the manufacturer’s instructions. The quality of RNA samples was determined using a 2100 Bioanalyzer (Agilent Technologies). For each sample, at least 20 µg of total RNA was sent to Beijing Genomics Institute for Illumina sequencing (commercial service). For qRT-PCR experiments, total RNA was isolated from the roots of control and treated (500 mM NaCl for varying time periods; 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h) greenhouse-grown A. officinalis and control and treated (150 mM NaCl for varying time periods; 0 h, 1 h, 3 h, 6 h and 24 h) roots of one-week-old Arabidopsis seedlings as described above. An aliquot of this RNA (1 µg) was used to synthesize cDNA using Maxima first strand cDNA synthesis kit for qRT-PCR (Thermo Scientific) following manufacturer’s instructions.
8
DNA Extraction and Sanger Sequencing Protocol
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DNA was extracted using a DNAeasy blood and tissue kit (Qiagen), and target DNA was amplified using LAtaq (TakaraBio) according to the protocol provided by the manufacturer. For analysis of the transcript, RNA was extracted using an ISOLATE II RNA Mini Kit (Meridian Bioscience, Cincinnati, OH). Extracted RNA was reverse transcribed using a Maxima First Strand cDNA Synthesis Kit for qRT-PCR (Thermo Fisher Scientific). Primers for the amplification of SLC10A target site are listed in Table S1 . Sanger sequencing was performed after gel purification using NucleoSpin gel and a PCR Clean-up kit (Macherey-Nagel, Dueren, Germany). Base editing efficacy was estimated using EditR (http://baseeditr.com/ ), an algorithm for predicting potential editing in a guide RNA region from a single Sanger sequencing run.38 For the genotyping assay, the S267F variant (rs2296651) was genotyped using a commercial TaqMan SNP Genotyping Assay (Assay ID: C__16,184,554_10; Applied Biosystems, Foster City, CA) with TaqMan Genotyping Master Mix (Applied Biosystems) on the ViiA 7 Real-Time PCR System (Applied Biosystems) according to the manufacturer's protocol.
9
Quantitative RT-PCR Analysis of ANGPTL4 and PDK4 in hBM-MSCs
10
Comprehensive RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from leaf and root tissues of control and treated (500 mM NaCl for varying time periods; 0, 1, 3, 6, 12, and 24 h) greenhouse-grown A. officinalis and control and treated (50 mM NaCl for varying time periods; 0, 1, 3, 6, 12, and 24 h) tissues of 1-week-old WT Arabidopsis seedlings using TRIzol™ reagent (Life Technologies) following the manufacturer’s instructions. An aliquot of this RNA (1 µg) was used to synthesize cDNA using Maxima first strand cDNA synthesis kit for qRT-PCR (Thermo Scientific) following the manufacturer’s instructions. For genotyping and expression analysis of mutants and the heterologous expression lines, DNA and RNA were extracted from leaves of four-week-old seedlings. The qRT-PCR for selected genes was performed as described earlier (Krishnamurthy et al., 2019 (link)). The primers used in the study are listed in Supplementary Table S1
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