Trizol (Invitrogen, America) was used to extract total cellular RNA following the manufacturer's instructions. Quality and quantity of RNA were determined using Nanodrop One (ThermoFisher, America) at 260 nm and 260/280 nm, respectively. One μg of total RNA was reverse transcribed to obtain cDNA using RevertAid First Strand cDNA Synthesis Kit (Invitrogen, USA) in the T100TM Thermal Cycler (Bio-rad, America). Quantitative gene expression was performed following the FastStart Universal SYBR@ Green Master (Invitrogen, America) protocol using a CFX96TM Real-Time PCR System (Bio-rad, America). Primer sequences used (Takara, Japan) for qRT-PCR are presented in Table 1 . qRT-PCR data was analyzed using the 2−ΔΔCt method with threshold cycle values.
Faststart universal sybr green master
Manufactured by Thermo Fisher Scientific
Sourced in United States
FastStart Universal SYBR Green Master is a ready-to-use qPCR master mix that contains all the necessary components for real-time PCR amplification and detection with SYBR Green I dye.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Nephrin, by Boster Bio (1 mentions)
Streptozocin stz, by Merck Group (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
16 protocols using faststart universal sybr green master
1
Quantitative Gene Expression Analysis
2
Quantitative Analysis of mRNA and miRNA
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Total RNAs from tissues and cells were extracted using Trizol (Invitrogen, Carlsbad, CA, USA) accordint to the manufacturer’s protocols. 1 μg of total RNA was subjected to reverse transcription of mRNAs using dT18 as primer and reverse transcription kit (TakaRa Biotechnology (Dalian) Co., Ltd. Dalian, Liaoning, China) to generate total cDNA. Then the mRNA quantitative PCR was carried using primers in Table 2 and FastStart Universal SYBR Green Master (Invitrogen, Carlsbad, CA, USA) using StepOneTM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Gapdh was used for normalization. The reverse transcription and quantitative PCR of miRNA were carried using special probes for miR-98 and U6 (Applied Biosystems, Foster City, CA, USA) with TaqMan MicroRNA reverse transcription kit and Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The quantification was normalized to an endogenous control U6. Each sample in each group was detected in triplicate. The experiment was repeated at least three times.
3
Streptozocin-Induced Podocyte Apoptosis
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The main materials used in this study were streptozocin (STZ) (Sigma-Aldrich, St Louis, MO, USA); Cell Counting Kit-8 (CCK-8) and JC-1 mitochondrial membrane potential assay kit (Beyotime, Shanghai, China); Lipofectamine 2000 reagent (GenePharma, Shanghai, China); antibodies against swiprosin-1 and podocin (Santa Cruz, Dallas, TX, USA); nephrin (Boster, Wuhan, China); cleaved caspase-8，Wilms Tumor 1 (WT-1, Novus biochemisry, Colorado, America); cleaved caspase-3 (Abcam, Cambridge, UK); cleaved caspase-9, Bax, Bcl-2, cytochrome C, total p38 and phospho-p38 (Cell Signaling Technology, Boston, MA, USA); tubulin (Beyotime, Shanghai, China); SB203580(Selleck Chemicals, Texas, America); BCA protein quantification kit (Bi-yun-tian, Shanghai); Trizol reagent, PrimeScript RT Master Mix Perfect Real Time kit and FastStart Universal SYBR Green Master (Invitrogen, Carlsbad, CA, USA); type I collagen (Biochrom, Berlin, Germany); mouse recombinant interferon (IFN-γ) (Immugenex, Los Angeles, CA); RPMI 1640, fetal bovine serum (FBS) (GIBCO, Grand Island, NY); mannitol (Beijing Dingguo Changsheng Biotechnology, Beijing, China). All chemicals were purchased from Sigma-Aldrich.
4
Formononetin, Metformin, and Carboxymethyl Cellulose Sodium Evaluation
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Formononetin (FMN, Purity: HPLC>98%), Metformin (MET), and carboxymethyl cellulose sodium (CMC-Na) were provided by Weikeqi Biotechnology Co., Ltd. (Sichuan, China). RevertAid First Strand cDNA Synthesis Kit (#K1622) and FastStart Universal SYBR Green Master (Rox) were purchased from Thermo Fisher Scientific Co., Ltd. (USA). All test assay kits and the SOD, MDA, GSH-Px, and CAT ELISA kits were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The full wavelength enzyme marker was provided by Thermo Fisher Scientific Co., Ltd. (USA). Hematoxylin-eosin stain (H&E), Periodic Acid-Schiff stain (PAS), and Masson staining kits were provided by Servicebio Company (Wuhan, China). Goat anti-Rabbit IgG and Goat anti-Mouse IgG were provided by Servicebio Company (Wuhan, China). The FMN was dissolved with 0.5% CMC-Na. The MET was dissolved with water.
5
Quantitative Real-Time PCR of Lung RNA
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RNA is extracted from homogenized lung tissues using TRIzol™ Reagent (ThermoFisher #15596026) according to the manufacturer’s protocol. Then RNA is quantified by Nanodrop 1000 (ThermoFisher) and reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher #4368814). FastStart Universal SYBR Green Master (ThermoFisher # 4913850001) was used for relative quantification of cDNA on the ViiA 7 Real-Time PCR System (ThermoFisher) (Primer information included in Table S3 ).
6
THP-1 Cytokine Expression Analysis
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THP-1 cells were pretreated with PT (10 μM)
for 2 h and stimulated by LPS (1 μg/mL) for 24 h. THP-1 cells
were subsequently mixed with NecleoZOL to isolate RNA. Total RNA was
quantified using a NanoDropTW 1000 Spectrophotometer (Thermo Fisher
Scientific, MA, USA). A specific amount of RNA was used to synthesize
cDNA with the MMLV reverse transcription kit (Thermo Fisher Scientific,
Catalog No. 28025013, MA, USA). RT-qPCR was conducted using a FastStart
Universal SYBR Green Master in the ABI Step One Plus Real-Time PCR
System (Thermo Fisher Scientific, MA, USA). Primers were purchased
from Genomics (Taipei, Taiwan), and their details are described inTable 1 .
for 2 h and stimulated by LPS (1 μg/mL) for 24 h. THP-1 cells
were subsequently mixed with NecleoZOL to isolate RNA. Total RNA was
quantified using a NanoDropTW 1000 Spectrophotometer (Thermo Fisher
Scientific, MA, USA). A specific amount of RNA was used to synthesize
cDNA with the MMLV reverse transcription kit (Thermo Fisher Scientific,
Catalog No. 28025013, MA, USA). RT-qPCR was conducted using a FastStart
Universal SYBR Green Master in the ABI Step One Plus Real-Time PCR
System (Thermo Fisher Scientific, MA, USA). Primers were purchased
from Genomics (Taipei, Taiwan), and their details are described in
7
Quantitative PCR Analysis of Mouse Ovarian Gene Expression
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The RNeasy Mini Kits (Qiagen, Germany) were used to extract RNA from mouse ovaries based on the manufacturer’s protocols. To generate cDNA, 1 μg of RNA with oligo dT and a reverse transcription kit (Transgen Biotech, CN) was used. The primers are presented in Table 1 , and FastStart Universal SYBR Green Master (Thermo Fisher Scientific, USA) were used to carry quantitative PCR with the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, USA). GAPDH was used for control. Each sample was tested three times [3 ].
Sequences of the primers used in QRT-PCR
| Target gene | Primer | Nucleotide sequence |
|---|---|---|
| h-GAPDH | F | 5′-GGAGCGAGATCCCTCCAAAAT-3′ |
| R | 5′-GGCTGTTGTCATACTTCTCATGG-3′ | |
| h-HO-1 | F | 5′-AAGACTGCGTTCCTGCTCAAC-3′ |
| R | 5′-AAAGCCCTACAGCAACTGTCG-3′ | |
| m-GAPDH | F | 5′-AGGTCGGTGAACGGATTTG-3′ |
| R | 5′-TGTAGACCATGTAGTTGAGGTCA-3′ | |
| m-BCL-2 | F | 5′-GCTACCGTCGTGACTTCGC-3′ |
| R | 5′-CCCCACCGAACTCAAAGAAGG-3′ | |
| m-Beclin1 | F | 5′-ATGGAGGGGTCTAAGGCGTC-3′ |
| R | 5′-TCCTCTCCTGAGTTAGCCTCT-3′ | |
| m-p62 | F | 5′-AGGATGGGGACTTGGTTGC-3′ |
| R | 5′-TCACAGATCACATTGGGGTGC-3′ | |
| m-LC3I/II | F | 5′-GACCGCTGTAAGGAGGTGC-3′ |
| R | 5′-CTTGACCAACTCGCTCATGTTA-3′ | |
| m-Atg5 | F | 5′-TGTGCTTCGAGATGTGTGGTT-3′ |
| R | 5′-GTCAAATAGCTGACTCTTGGCAA-3′ | |
| m-HO-1 | F | 5′-AAGCCGAGAATGCTGAGTTCA-3′ |
| R | 5′-GCCGTGTAGATATGGTACAAGGA-3′ |
8
Quantitative Analysis of CD54 Expression
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Total RNA was extracted from EC and paracancer tissues using TRIzol reagent (Thermo Fisher Scientific, Inc). Then, 5 µg RNA was reverse transcribed to complementary DNA (cDNA) using a RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Inc). qPCR was performed on an ABI 7500 Real-time PCR system (Thermo Fisher Scientific, Inc) using FastStart Universal SYBR-Green Master (Thermo Fisher Scientific, Inc), following the protocol. The thermocycling conditions were as follows: 95°C denaturation for 15 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The relative mRNA expression levels of target genes were normalized to β-actin and calculated using the 2-ΔΔCt method. The specific sequences of primers were as follows: CD54 forward: 5'-ATGCCCAGACATCTGTGTCC -3', reverse: 5'-GGGGTCTCTATGCCCAACAA -3'; β-actin forward: 5'-GTGGACATCCGCAAAGAC -3', reverse: 5'-GAAAGGGTGTAACGCAACT -3'. All experiments were performed in triplicate.
9
RNA Extraction and qRT-PCR Analysis
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RNA was extracted from the harvested cells using TRIzol™ Reagent (ThermoFisher #15596026) according to the manufacturer’s protocol. Then RNA was quantified by Nanodrop 1000 (ThermoFisher) and reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher #4368814). FastStart Universal SYBR Green Master (ThermoFisher #4913850001) was used for relative quantification of cDNA on the ViiA 7 Real-Time PCR System (ThermoFisher) (Primer information included in Supplementary Table 2 ).
10
RNA Extraction, Reverse Transcription, and qPCR for Gene Expression
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