Total RNA was isolated from NP cells using TRIzol reagent (Invitrogen, Waltham, MA, USA). First-strand cDNA was synthesized using the SuperScript system (Invitrogen, Waltham, MA, USA). PCR amplification conditions were as follows: denaturation at 94 °C for 20 s, annealing at 55 °C for 30 s, and extension at 72 °C for all genes. PCR reactions were performed using the QuantiTech SYBR Green PCR kit (Qiagen, Valencia, CA, USA) and an Exicycler 96 system (Bioneer, Daejeon, Korea). Specific primers for the genes examined were based on their PrimerBank sequences and are listed in Table 1 .
Exicycler 96 system
Manufactured by Bioneer
Sourced in United States, China
The Exicycler 96 system is a real-time PCR instrument designed for sensitive and accurate gene expression analysis. It features a 96-well format and supports fast, high-throughput sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green, by Solarbio (4 mentions)
Tripure, by BioTeke (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Quantitech sybr green pcr kit, by Qiagen (1 mentions)
Reverse transcription kit, by BioTeke (1 mentions)
Tripure reagent, by BioTeke (1 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Prime q mastermix, by GeNet Bio (1 mentions)
M mlv reverse transcriptase, by Enzynomics (1 mentions)
Tripure, by Tiangen Biotech (1 mentions)
Super m mlv reverse transcriptase, by Tiangen Biotech (1 mentions)
Power taq pcr mastermix, by Tiangen Biotech (1 mentions)
Beyort 2 m mlv reverse transcriptase, by Beyotime (1 mentions)
Sybr green reagent, by Solarbio (1 mentions)
Total rna isolation kit, by Tiangen Biotech (1 mentions)
Mirna first strand cdna synthesis tailing reaction, by Sangon (1 mentions)
High purity total rna rapid extraction kit, by BioTeke (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Taq hs perfect mix, by Takara Bio (1 mentions)
Power taq pcr mastermix, by BioTeke (1 mentions)
10 protocols using exicycler 96 system
1
Quantitative Real-Time PCR Protocol
2
Quantifying linc01224 and IGF2BP3 Expression
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Total RNA from HSCC cells was obtained using TRIpure reagent (BioTeke, Beijing, China) according to the manufacturer's protocol. A NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was applied to determine RNA quantity and quality. RNA was reverse transcribed into cDNA with the Reverse Transcription Kit (BioTeke). Quantitative real-time PCR (qRT-PCR) was performed with SYBR Green (Solarbio, Beijing, China) and an Exicycler 96 system (BIONEER, Daejeon, South Korea). The levels of linc01224 and IGF2BP3 expression were normalized to that of β-actin. Samples were analyzed in triplicate. The primers of forward and reverse sequences were as follows: linc01224, 5'-GAGCCAGGCACCCGTTTA-3' and 5'-GGTTGACAAGTGGTGGAATCTG-3'; IGF2BP3: 5'-TCGGAACATCACCAAACA-3' and 5'-GGTGCCTTCAGGAGTAGAG-3'; and β-actin: 5'-CTTAGTTGCGTTACACCCTTTCTTG-3' and 5'-CTGTCACCTTCACCGTTCCAGTTT-3'. The relative expression of linc01224 was calculated by the 2-△△CT method.
3
Comparative qRT-PCR Analysis of Lung Cell Genes
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Total RNA was extracted from lung tissues and BEAS-2B cells by TRIpure (BioTeke). The RNA was reverse transcribed into cDNA using BeyoRT™ II M-MLV RNase H- (Beyotime). Real-time PCR assay was conducted on Exicycler 96 system (BIONEER) using SYBR Green (Solarbio). The results was calculated using 2-△△CT method. The primer sequences are as follows. Mus musculus ACPL2: F, AATCGCTTCTTGGTGCTG; R, CTACGCTTGGAATGTTGC. Mus musculus RABL4: F, GAAATGCTGGATAAGTTGTG; R, GAGGGAGATGCCTGAAGT. Mus musculus SLC27A3: F, GCATTGTGGGCTGCTTGG; R, GGGCTGGTTGACGAGGTAT. Mus musculus STAU1 F, GTAAAGAAACCAGGAGACG; R, CTGCTGATGGCTAAGATAA.
Mus musculus β-actin: F, CTGTGCCCATCTACGAGGGCTAT; R, TTTGATGTCACGCACGATTTCC. Homo sapiens ACPL2: F, ATGGAGCACTTCAAGGTAA; R, AGCAGAGTAGAGGGCAAA. Homo sapiens RABL4: F, GGAATGGATTTGGTGGTGAA; R, GAGATGCCTGGAGCCTGTGA. Homo sapiens SLC27A3: F, GTTCGGATGGCAAATGAGG; R, TGTACCGGGCAGTTGTGAG. Homo sapiens STAU1 F, ATCCGATTAGCCGACTGG; R, ACTTGAGTGCGGGTTTGG. Homo sapiens β-actin: F, GGCACCCAGCACAATGAA; R, TAGAAGCATTTGCGGTGG.
Mus musculus β-actin: F, CTGTGCCCATCTACGAGGGCTAT; R, TTTGATGTCACGCACGATTTCC. Homo sapiens ACPL2: F, ATGGAGCACTTCAAGGTAA; R, AGCAGAGTAGAGGGCAAA. Homo sapiens RABL4: F, GGAATGGATTTGGTGGTGAA; R, GAGATGCCTGGAGCCTGTGA. Homo sapiens SLC27A3: F, GTTCGGATGGCAAATGAGG; R, TGTACCGGGCAGTTGTGAG. Homo sapiens STAU1 F, ATCCGATTAGCCGACTGG; R, ACTTGAGTGCGGGTTTGG. Homo sapiens β-actin: F, GGCACCCAGCACAATGAA; R, TAGAAGCATTTGCGGTGG.
4
Quantitative RT-PCR Analysis of Gene Expression
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RNA extraction and DNase treatment were performed as described above for RNA-seq. cDNA was synthesized using M-MLV reverse transcriptase (Enzynomics) with specific primers for semi-quantitative RT-PCR. qRT-PCR was performed on an Exicycler 96 system (Bioneer) using Prime Q-master mix (Genet Bio). Expression levels of each mRNA of interest were normalized to that of the gapA gene50 (link). All experiments were performed according to the manufacturer’s instructions.
5
Quantification of miR-19b-3p expression
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The total RNA was extracted from femur tissues at the indicated time or differentiated BMSCs using TRIpure (TianGEN, China) and transcribed into the relevant cDNA by the Super MMLV Reverse Transcriptase (TianGEN, China). Stem‐loop RT‐PCR was used to quantify miR‐19b‐3p. RT‐PCR conducted in Exicycler 96 System (Bioneer, Korea) using 2 × Power Taq PCR MasterMix (TianGEN, China) and SYBR Green (Solarbio, China). The levels of miR‐19b‐3p were presented as relative expression, which calculated by comparing with the control group. Primers were as follows:
rno‐miR‐19b‐3p forward: 5'‐TGTGCAAATCCATGCAAAACTGA‐3';
reverse: 5'‐GCAGGGTCCGAGGTATTC‐3';
rno‐miR‐19b‐3p forward: 5'‐TGTGCAAATCCATGCAAAACTGA‐3';
reverse: 5'‐GCAGGGTCCGAGGTATTC‐3';
6
Gene Expression Analysis of Colon Tissues
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Total RNA from the ground colon tissues in nitrogen and cultured cells was extracted using the TRIpure Reagent lysis buffer (RP1001, BioTeke, China), followed by cDNAs reverse transcription with the BeyoRT II M-MLV reverse transcriptase (D7160L, Beyotime). qPCR was performed with the SYBR Green reagent (SY1020, Solarbio) on the Exicycler 96 system (Bioneer, Korea). The primer sequences (5’-3’) were shown: Nfatc3 forward GGTAAAGAGCAGCACATA, Nfatc3 reverse TTGACTAGAGGCAGGATT; MCP-1 forward GCCTGCTGTTCACAGTTGCC, MCP-1 reverse CTGGACCCATTCCTTCTTGG; TNF-α forward CAGGCGGTGCCTATGTCTCA, TNF-α reverse GCTCCTCCACTTGGTGGTTT; IL-6 forward ATGGCAATTCTGATTGTATG, IL-6 reverse GACTCTGGCTTTGTCTTTCT; IL-1β forward CTCAACTGTGAAATGCCACC, IL-1β reverse GAGTGATACTGCCTGCCTGA; Pou3f1 forward CGTGTTCTCGCAGACCACCATC, Pou3f1 reverse CGCACCACCTCCTTCTCCAGTT; GAPDH forward TGTTCCTACCCCCAATGTGTCCGTC, GAPDH reverse CTGGTCCTCAGTGTAGCCCAAGATG. The relative gene expression was calculated with the 2−ΔΔCt method and normalized to GAPDH.
7
Quantification of miR-378a-5p Expression
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The total RNA of clinical tissue specimens was extracted using total RNA isolation kit (Tiangen, China) to synthesize cDNA in accordance with the manufacturer’s instruction of miRNA First Strand cDNA Synthesis (Tailing Reaction) (Sangon Biotech, Shanghai, China). The expression level of miR-378a-5p in the clinical samples was determined by the Exicycler 96 System (Bioneer, Korea) in the present of Power Taq PCR MasterMix (Solarbio, China) and SYB Green (Solarbio, China). MiR-378a-5p expression was analyzed by 2−ΔCT method.
8
miRNA-34a and Klf4 Expression Analysis
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The total RNA in the perihemorrhagic penumbra was extracted using the high purity total RNA rapid extraction kit (BioTeke, Beijng, China) and quantified by an ultraviolet spectrophotometer. The isolated RNA was transcribed into the relevant cDNA by miR-34a-5p stem-loop RT primer or respective primer, the expression levels of miR-34a-5p and Klf4 were detected by RT-PCR utilizing Taq HS Perfect Mix (Takara, Dalian, China) and SYBR Green (Takara) in an Exicycler 96 System (Bioneer, Daejeon, Korea). 5S and β-actin were used as the internal reference. The data was analyzed by 2-ΔΔCT method.
The primers for RT-PCR were as follows:
MiR-34a-5p stem-loop RT primer: 5’-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACAACC-3’; Forward: 5’-GGACTTGGCAGTGTCTTAGCTG-3’; Reverse: 5’-GTGCAGGGTCCGAGGTATTC-3’
Klf4 Forward: 5’-GGAGCCCAAGCCAAAGAGG-3’; Reverse: 5’-CGTCCCAGTCACAGTGGTAAGGT-3’
The primers for RT-PCR were as follows:
MiR-34a-5p stem-loop RT primer: 5’-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACAACC-3’; Forward: 5’-GGACTTGGCAGTGTCTTAGCTG-3’; Reverse: 5’-GTGCAGGGTCCGAGGTATTC-3’
Klf4 Forward: 5’-GGAGCCCAAGCCAAAGAGG-3’; Reverse: 5’-CGTCCCAGTCACAGTGGTAAGGT-3’
9
Quantitative Analysis of SPRED1 Expression
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RNA was extracted from cells of bone marrow samples and cell lines using the TRIpure (BioTeke, Wuxi, China) and reverse transcribed using a Super M-MLV Reverse Transcription Reagent Kit (BioTeke) according to the manufacturers’ protocol. Quantitative reverse transcription PCR (RT-PCR) was performed using 2× Power Taq PCR MasterMix (BioTeke) and SYBR Green (Solarbio, Beijing, China) as a double-stranded DNA-specific dye. Specific primer sequences of SPRED1 designed by Sangon Biotech (Shanghai, China) were as follows: forward: 5′-TTTTCTGATCCCTGTTCGTG-3′ and reverse: 5′-TCCAGCAGCTTTATGTTTCC-3′. The following steps of RT-PCR followed protocols in our laboratory. The relative level of SPRED1 was analyzed using the Exicycler™ 96 System (BIONEER, Daejeon, Korea) and calculated using the 2−ΔΔCt method.
10
Quantitative Analysis of HPV 16/18
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All samples with inconsistent results were further analysed using qPCR. The set of primers and probes for E2/E6 of HPV16 and HPV18 were designed according to previous studies (16, 17) . qPCR was conducted using an Exicycler 96 system (Bioneer Corporation), as previously described (16, 17) , and 10 µl of the clinical samples were used as the DNA templates. The standard curves were obtained from amplification of a dilution series using 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 and 10 1 copies of HPV16 and HPV18 plasmid DNA.
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