Total RNA was extracted from Formalin-Fixed, Paraffin-Embedded tissue (FFPE) using PureLink RNA Mini Kit (Thermo Fisher). First strand cDNA was synthesized using 5X All-In-On MasterMix (MasterMix-LR, Diamed) per manufacturer’s protocol. Reverse transcriptase real-time PCR (RT-PCR) was carried out on 96-well plates using iTaq Universal SYBR Green Supermix (BioRad). Concentrations for each sample were measured using the NanoDrop ND-100 spectrophotometer 119 (NanoDrop Technologies, Wilmington, DE, USA) and Qubit (Thermo Fischer Scientific, 120 Waltham, MA, USA). The primer sequences were designed using Primer Express™ Software v3.0.1 (ThermoFisher Scientific, USA) (Supplemental material).
Primer express software v3
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Primer Express Software v3.0 is a tool designed for the design of TaqMan and SYBR Green primers and probes for real-time PCR experiments. The software provides algorithms for optimal primer and probe selection based on user-provided input sequences.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Rneasy mini kit, by Qiagen (9 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Sybr green pcr assay, by Thermo Fisher Scientific (5 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Tri reagent, by Molecular Research Center (4 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (3 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Superase in, by Thermo Fisher Scientific (3 mentions)
Rnase free dnase, by Qiagen (3 mentions)
Cfx real time system, by Bio-Rad (3 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (3 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
80 protocols using primer express software v3
1
FFPE RNA Extraction and RT-PCR
2
Validating Microarray Findings with qPCR
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Microarray results were validated with real-time quantitative polymerase chain reaction (qPCR), which was performed using the same samples (four individual/replicates per clone) used in the array. Validation was performed comparing microarray normalized fluoresce values with normalized qPCR mRNA abundance, both log e transformed. We selected 15 differentially expressed genes belonging to the tryptophan/serotonergic synapse (tryptophan 2,3-dioxygenase A, TDO2; kynurenine formamidase, Kyn; dopamine decarboxylase, DDC; serotonin transporter, SERT; G protein subunit alpha q, Gq;, arachidonic acid (prostaglandin E2 receptor EP4 subtype, PTGER4; prostaglandin-endoperoxide synthase 1, PTGS1 or COX1; cyclooxygenase-like facilitator of follicle, PXT; Prostamide/prostaglandin F synthase, PGFS; Prostaglandin reductase, PTGR1; Prostaglandin E synthase 3,PTGE3) and insulin growth factor signaling pathways (Insulin like peptide, ILP; Insulin receptor, IR; phosphatidylinositol 3-kinase, PI3-kp85/p60 and Forkhead transcriptional factor FOXO10 (link),18 ,22 ,27 ,30 (link),35 (link)). The G3PDH gene (glyceraldehyde 3-phosphate dehydrogenase) was used as an internal control (house-keeping gene). Primers for each one of these genes were designed with Primer Express® Software v3.0.1(Thermofisher, USA) and are provided in Table S1 . qPCR was performed according to manufacturer’s protocols.
3
RNA Isolation and Gene Expression Analysis
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The total RNA from collected hydrogels was isolated using the GeneJET RNA Purification Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Gene expressions were then analyzed with SYBR Green PCR Master Mix and StepOnePlus real-time PCR system (Applied Biosystems). The fold changes of the target genes were calculated using the 2–ΔΔCt method by normalizing them with the housekeeping gene (GAPDH) expression. Coding sequences for GAPDH, matrix metalloproteinase-2 (MMP2), catenin beta-1 (β-catenin), and integrin beta-1 (integrin β1) were designed using the National Center for Biotechnology Information reference sequences (Table 1 ) and Primer Express software v3.0.1 (Thermo Fisher Scientific) for preparing primers.
4
Quantifying Gene Expression in Rat Pulmonary Vessels
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Total RNA was isolated from rat distal pulmonary vessels and PASMCs using a TRIzol™ reagent (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. RNA purity was measured as the A260/A280 ratio using a Multiskan Sky Microplate spectrophotometer (cat. no. 51119570; Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1,000 ng) was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). Reverse transcription was performed using the following temperature protocol: 37°C for 1 h and 94°C for 5 min. All primers used were designed using the Primer Express™ software v.3.0.1 (Thermo Fisher Scientific, Inc.) and are listed in Table SI . The reverse-transcribed cDNA was then subjected to PCR using Taq DNA polymerase (cat. no. 10342020; Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, qPCR was performed using the SYBR-GreenER PCR kit. The following thermocycling conditions were used for qPCR: Initial denaturation at 94°C for 20 sec; followed by 45 cycles of 60°C for 30 sec and 72°C for 60 sec; and a cooling step at 4°C. mRNA expression levels were quantified using the 2−ΔΔCq method (25 (link)) and normalized to the internal reference gene GAPDH.
5
Validating Microarray with qPCR
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Microarray results were validated with real-time quantitative polymerase chain reaction (qPCR). We selected four differentially expressed genes from different pathways/gene families which had already been successfully used in previous studies [46 (link), 67 (link)]: Krebs cycle (isocytrate dehydrogenase, idh; ATP citrate lyase, ATPCL), tryptophan metabolism (dopamine decarboxylase, Ddc) and Ecdysteroid pathways (Ecdysone receptor, EcR b). The G3PDH gene (glyceraldehyde 3-phosphate dehydrogenase) was used as internal control (Housekeeping). Primers for each one of these genes were designed with Primer Express® Software v3.0.1(Thermofisher, USA) and are provided elsewhere [46 (link), 67 (link)]. qPCR was performed according to manufacturer’s protocols.
6
RNA Extraction and Gene Expression Analysis
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Pre-culture was conducted using 5 ml of TSB, initiated using a single colony from an agar plate, in a shaking incubator at 37°C, overnight. Cells were cultured using 5 ml TSB with 1% inoculum in a shaking incubator at 37°C for 12 and 24 h to extract total RNA. Cells were harvested by centrifugation at 3,521 ×g for 20 min. Then, total RNA was prepared using the TRIzol Reagent and reverse transcription was performed using Superscript IV Reverse Transcriptase (Invitrogen Co., USA) to generate cDNA, as per the manufacturer’s instructions. Primers were designed using Primer express software v3.0.1 from Thermo Fisher Scientific (USA) (Table S1 ). These primers generated amplicons of 150 bp when comparing gene expression. Prior to semi-quantitative PCR, the cycle number was optimized to determine the saturated gene expression levels of DNA gyrase subunit B (gyrB, the endogenous control) for each template. After optimization, the optimal cycle number was set at 25 cycles to enable the further comparative analysis of gene expression. Semi-quantitative PCR was conducted using LA taq with GC buffer I (Takara Medical Co., Ltd.), using the methods in the manual.
7
TRIzol-based RNA Extraction and qPCR Analysis
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In this work, we applied TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in cell lysis. After extraction, Revert Aid First Strand cDNA Synthesis Kit (Takara, Tokyo, Japan) was used to synthesize cDNA from total RNA through the reverse transcription in line with specific instructions. The complementary DNA served as the template to perform PCR. Results were then detected by a LightCycler ® 480 thermal cycler. Conditions for thermal cycling included, 5-min initial denaturation under 95 °C; 10 s under 95 °C, 20 s under 65 °C, and 30 s under 72 °C for altogether 45 cycles. Table 2 displays sequences of all primers prepared with the Primer Express Software v3.0.1 (Thermo Fisher Scientific, Inc.). Gapdh served as the reference for normalizing gene expression.
8
Hippocampal RNA Extraction and qPCR Analysis
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Hippocampal tissue RNA extraction was executed using the Hybrid-R kit (305-010; GeneAll Biotechnology, Seoul, South Korea). RNA concentration was measured using a spectrophotometer (Nano Drop ND-1000, NanoDrop Technologies Inc.). cDNA was prepared from 1 μg of total RNA using the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, Shiga, Japan). PCR amplification was executed using the SYBR-Green reagent (Takara Bio) in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed in 20 μL reaction volumes. Sequences for oligonucleotide primers were selected using the Gene Database of National Center for Biotechnology Information (NCBI) and Primer Express™ Software v3.0.1 (Thermo Fisher Scientific, Waltham, MA, USA). Primer pairs are listed in Table 3 and were verified using a melting curve analysis (Supplementary Materials S3 ).
9
Quantitative PCR Analysis Protocol
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A qPCR analysis was performed as described previously [9 (link)]. The primers used for qPCR were designed by Primer Express® Software v3.0.1 (Thermo Fisher, Waltham, MA, USA). The primer sequence is indicated in Supplementary Table S4 . Results are representative of at least three independent experiments.
10
RNA Extraction and Real-Time PCR Analysis
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RNA was extracted by using the Hybrid-R kit (305-010; GeneAll Biotechnology, Seoul, Korea). The concentration of RNA was measured using a spectrophotometer (Nano Drop ND-1000, NanoDrop Technologies Inc). cDNA was prepared from 1 μg of total RNA using the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, Shiga, Japan). PCR amplification was executed using the SYBR-Green reagent (Takara Bio) in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed in 20 μL reaction volumes. Sequences for oligonucleotide primers were selected using the Gene Database of National Center for Biotechnology Information (NCBI) and Primer Express™ Software v3.0.1 (Thermo Fisher Scientific, Waltham, MA, USA). Primer pairs are listed in Table 1 .
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