HRMECs were lysed with TRIzol (Invitrogen, Carlsbad, CA, USA), and total RNA was extracted according to the manufacturer's protocol. The concentration and integrity of total RNA were detected with UV spectrophotometry (NANODROP 2000C, Thermo, USA). Reverse transcriptase reactions were performed using a Thermo Revert Aid TM First Strand cDNA Synthesis Kit (K1622, Thermo, USA). Real-time PCR reactions were performed with 2 × SYBR Select Master Mix (Cat. number 4472908, Invitrogen, USA) using a real-time PCR system (Piko Real 96 PCR system, Thermo Scientific, USA). Each sample was measured in triplicate wells. The target gene primers are shown in Table 2 . Data were normalized to the housekeeping genes human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We calculated the changes in mRNA expression according to the 2−ΔΔCT method, with ΔCT = CTTarget gene − CTGAPDH and ΔΔCT = ΔCTTreatment − ΔCTControl. Each experiment was repeated at least three times.
Piko real 96 pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Piko Real 96 PCR system is a high-performance real-time PCR instrument designed for efficient nucleic acid amplification and detection. It features a 96-well format and supports a wide range of applications, including gene expression analysis, pathogen detection, and genotyping.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (3 mentions)
Sybr select master mix, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Thermo revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Novostart sybr qpcr supermix plus kit, by Novoprotein (1 mentions)
Ultra sybr mixture with rox, by CWBIO (1 mentions)
Mirna real time pcr assay kit, by CWBIO (1 mentions)
Sybr green pcr kit, by Roche (1 mentions)
Easyscript first strand cdna synthesis supermix, by Transgene (1 mentions)
Pikoreal 2, by Thermo Fisher Scientific (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Ultrasybr mixture, by CoWin Biotech (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
8 protocols using piko real 96 pcr system
1
Quantitative Gene Expression Analysis
2
Quantitative Analysis of miR-503 and Genes
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In terms of the manufacturer's protocol, TRIzol (Invitrogen, Carlsbad, CA, USA) was added to the HMEC-1 cells for lysis and total RNA was extracted. Total RNA concentration and integrity were determined by UV spectrophotometry (NANODROP 2000C, Thermo, USA). The reverse transcriptase reaction was carried out using a Thermo Revert AidTM First Strand cDNA Synthesis Kit (K1622, Thermo, USA). qPCR reactions were performed using 2 × SYBR Select Master Mix (Invitrogen, USA) and a Real-time PCR system (Piko Real 96 PCR system, Thermo Scientific, USA). Each sample was measured in three wells. The data was normalized to the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6. The relative expression of miR-503 and mRNA of genes were calculated and quantified using 2−ΔΔCt method.
3
Quantification of Pericyte-derived Apelin mRNA
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Total RNA was isolated from cultured pericytes using Trizol reagent (Invitrogen, CA, US), and we determined the concentration and integrity of total RNA with UV spectrophotometry (NANODROP 2000C, Thermo, US). We used the Fermentas reverse transcription system (Fermentas, St. Leon-Rot, Germany) to reverse RNA (1 μg) into first strand cDNA, using a real-time PCR system (PikoReal 96 PCR system, Thermo Scientific). The PCR solution system contained 1 µL of cDNA (1 : 20 diluted), specific primers 1 µL (10 pmol), 3 µL DEPC-water, and 5 µL of SYBR Select Master Mix (Invitrogen), with a final volume of 10 µL. Each sample was measured in triplicate wells. Primers used were as follows: β-actin Forward: 5′-TGGCTCTATCCTGGCCTCACT-3′, β-actin Reverse: 5′-GCTCAGTAACAGTCCGCCTAGAA-3′; rat apelin Forward: 5′-GATGGAGAAAGGCGAAGAAAG-3′, rat apelin Reverse: 5′-GGTGAGAGATGAGACCACTTGT-3′. The standard PCR conditions included 2 min at 50°C and 10 min at 95°C, followed by 35 cycles of extension at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The mRNA expression was normalized to the expression level of ACTB. We calculated the changes in mRNA expression according to the 2−ΔΔCT method, with ΔCT = CTarget gene − CTACTB and ΔΔCT = ΔCTreatment − ΔCTControl. Each experiment was repeated at least three times.
4
Transcriptome Validation of P. lobata via qRT-PCR
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To validate the transcriptome data of P. lobata, a qRT-PCR was carried out on the PIKOREAL 96 PCR System (Thermo Scientific, Waltham, MA, USA) with the Novostart SYBR qPCR SuperMix Plus kit (Novoprotein, Shanghai, China). Primer pairs were designed to amplify the actin gene and nine unigenes involved in isoflavonoid biosynthesis using Primer Premier (v5.0) (Table S3 ). All reactions were prepared in a final volume of 10 µl (containing 1 µl of cDNA, 1 µl of each specific primer, 5 µL of 2 × SYBR Green mixture, and 2 µL of RNase-free water). The qRT-PCR was run under these conditions: 1 min at 95 °C, 40 cycles of 95 °C for 20 s, and 60 °C for 1 min. Relative expression levels were normalized to those of actin (CL10129.Contig5) mRNA, by applying the 2 −ΔΔCt method (Livak & Schmittgen, 2001 (link)). Every target gene was run in three biological replicates.
5
Quantifying miRNA Expression by RT-qPCR
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We obtained total miRNA from cells by using Trizol reagent (Invitrogen, USA) according to its illustration. RT-qPCR was carried out on a Pikoreal 96 PCR system (Thermo Fisher Scientific, USA). UltraSYBR Mixture (with ROX) (CWBio, China) and the miRNA Real-Time PCR Assay kit (CWBio, China.) were used to detect and measure relative expression of mRNA as well as miRNA, respectively. We normalized the results and analyzed the data based on the 2-ΔΔCt method. The primers in this study were indicated in Table 1 .
6
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was isolated using TRIzol reagent (15596026, Invitrogen, Waltham, MA, USA) and reverse-transcribed into cDNAs with EasyScript® First-Strand cDNA Synthesis SuperMix (AE301-02, TransGen Biotech, Beijing, China) according to the manufacturer’s protocols.
The cDNA templates were then subjected to RT–qPCR using the SYBR Green PCR Kit (04913914001, Roche, Mannheim, Germany) according to the manufacturer’s instructions. The assay was performed with a PikoReal 96 PCR System (Thermo Fisher Scientific, Waltham, MA, USA). β-Actin was used as an internal control. The relative expression levels were quantified and analyzed using PikoReal 2.1 software (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of the target genes were determined using the 2−ΔΔCt method. Each sample was analyzed in triplicate. The primers were all synthesized and purchased from GENEWIZ Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). The primer sequences are listed inSupplementary Table S2 .
The cDNA templates were then subjected to RT–qPCR using the SYBR Green PCR Kit (04913914001, Roche, Mannheim, Germany) according to the manufacturer’s instructions. The assay was performed with a PikoReal 96 PCR System (Thermo Fisher Scientific, Waltham, MA, USA). β-Actin was used as an internal control. The relative expression levels were quantified and analyzed using PikoReal 2.1 software (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of the target genes were determined using the 2−ΔΔCt method. Each sample was analyzed in triplicate. The primers were all synthesized and purchased from GENEWIZ Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). The primer sequences are listed in
7
Quantifying lncRNA and mRNA Expression
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Total RNA was extracted from lung tissues, and cells were cultured using Trizol reagent (Life Technologies Corporation, CA, USA). RNA was reverse transcribed using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). Then, real-time PCR was performed with UltraSYBR Mixture (ComWin Biotech Corporation, Beijing, China) following the manufacturer’s protocol in PIKO REAL 96 PCR System (Thermo Fisher Scientific, USA). The sequence of primers is shown in the Supplementary Material: Table S1
8
Quantitative gene expression analysis
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Total RNA was extracted from pellets or cells in each culture condition with Trizol Reagent (New Industry) following the manufacturer’s protocol. Total RNA was quantified with the Nanodrop Spectrophotometer (ND-1000, Thermo), and the reverse transcription reaction was performed on 1000 ng of RNA as previously described [13 (link)]. Quantitative real-time polymerase chain reactions (PCR) were performed on a Pikoreal 96 PCR System (Thermo) following the manufacturer’s procedures. The expression of type I collagen (COL I), type II collagen (COL II), type X collagen (COL X), aggrecan (ACAN), and SOX9 were analyzed with qRT-PCR with the gene-specific primers listed in Additional file 1 : Table. S1. The target genes of each sample were normalized to the values of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Relative expression of each gene was expressed as fold changes by the 2−ΔΔCt method. Five samples of each group were measured. Statistical significance was marked with different letters (P < 0.05).
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