At 3 months after CA induction, total RNAs from the whole Willis ring tissue were extracted by Trizol reagent (Life Technologies, Grand Island, NY, USA) in accordance with the manufacturer’s instructions. RT-PCR was performed using a Takara PrimeScript® RT reagent Kit (Takara, Dalian, China) and SYBR Green Realtime PCR Kit (QIAGEN, Hilden, Germany). Primer sequences were: forward 5′-ACGATCTGTTTCCCCTCATC-3′, reverse 5′-TGCTTCTCTCCCCAGGAATA-3′ for NF-κB (nuclear factor κB); 5′-TTCAGGTATGCGGTATTTGG-3′ and 5′-GTTGGAAGTGTAGCGTTTCG-3′ for iNOS (inducible nitric oxide synthase); 5′-AGCCGGGACTTCATCAATCAG-3′ and 5′-GCCCAAACACCAGCTCACTCTC-3′ for eNOS (endothelial nitric oxide synthase); forward 5′-CTGATAACCTGGATGCAGTCGT-3′, reverse 5′-CCAGCCAGTCCGATTTGA-3′ for MMP-2 (matrix metalloproteinase-2); forward 5′-TTCAAGGACGGTCGGTATT-3′, reverse 5′-CTCGAGCCTAGACCCAACTTA-3′ for MMP-9 (matrix metalloproteinase-9); forward 5′-AAGAAGGTGGTGAAGCAGGC-3′, reverse 5′-TCCACCACCCTGTT GCTGTA-3′ for GAPDH (internal control). The mRNA content was normalized to GAPDH mRNA level and expressed as fold change relative to control levels. All reactions were performed in triplicate.
Sybr green realtime pcr kit
Manufactured by Qiagen
Sourced in Germany, China
The SYBR Green Realtime PCR Kit is a laboratory equipment product designed for real-time polymerase chain reaction (PCR) analysis. It contains SYBR Green I dye, which binds to double-stranded DNA, allowing for the detection and quantification of DNA sequences during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Abi 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Superscript 2 first strand cdna synthesis system, by Thermo Fisher Scientific (1 mentions)
Mastercycler ep realplex, by Eppendorf (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Hairpin it mirna qpcr kit, by GenePharma (1 mentions)
Reverse transcript kit, by Takara Bio (1 mentions)
Hairpin it mirnas qpcr kit, by GenePharma (1 mentions)
7 protocols using sybr green realtime pcr kit
1
Expression Analysis of Cerebrovascular Markers
2
Quantitative Analysis of MMP-9 Expression
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Total RNA was isolated from DU145 cells and then quantitated by an ultraviolet spectrophotometer. Next cDNA was synthesized from RNA (200 ng) in a 20 µl reverse transcription reaction (Promega Corporation, Madison, WI, USA). qPCR was worked as follow: 30 sec at 94°C for denaturation, 30 sec at 54°C for annealing and 30 sec at 65°C for extension, for a total of 30 cycles. qPCR was completed with the SYBR green real-time PCR kit (Qiagen, Hilden, Germany) and the ABI 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR products were run on 2% agarose gels to prove that correct molecular sizes were present. Each sample was analyzed in triplicate using RT-qPCR. The primers for RT-qPCR as follow: MMP-9, forward 5′-AATCTCTTCTAGAGACTGGGAAGGAG-3′ and reverse 5′-AGCTGATTGACTAAAGTAGCTGGA-3′; GAPDH, forward 5′-AGAGAGAGGCCCTCAGTTGCT-3′ and reverse 5′-TTGTGAGGGAGATGCTCAGTGT-3′, synthesized by BGI Tech (Shenzhen Co., Ltd., Shenzhen, China). GAPDH was used as an internal control. Fold changes were analyzed using the 2−ΔΔCq method.
3
Quantitative Real-Time PCR Protocol
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Total RNA was extracted using TRIzol reagent (BY11909, Hefei Bomei Biotechnology Bo., Ltd., China), and Nanodrop (Thermo Scientific™, San Diego, CA, U.S.A.) was used to measure the concentration of RNA, which was then diluted to 500 ng/μl. Superscript II first-strand cDNA synthesis System (Invitrogen, U.S.A.) was used to determine reverse transcription. The mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR Green Real Time PCR kit (204057, QIAGEN, https://www.qiagen.com/cn/products/ , China). Next, 4 μl cDNA was mixed with 5 μl SYBR (codeDRR041A, Takara, Japan) and 1 μl Primer. PCR cycle was conducted as follows: pretreatment at 94°C for 2 min, at 94°C for 30 s, at 63°C for 30 s, at 72°C for 1 min (35 cycles), finally, chain extension at 72°C for 7 min and kept at 4°C. Sequences of primers used were listed in Table 1 . The expression levels of RT-PCR products were determined by the 2−ΔΔCT method [17 (link)].
4
Quantitative Real-Time PCR Analysis
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Real time PCR was carried out using SYBR green Real time PCR kit from Qiagen on an Eppendorf mastercycler, ep realplex (Eppendorf, Germany). Relative mRNA expression was determined by normalization to the expression of a housekeeping gene, beta-actin and U6 for microRNA gene expression. Primer list is provided in S1 Table .
5
Gene Expression Analysis by RT-qPCR
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The isolation of total RNA from transfected cells was processed with TRIzol reagent (Invitrogen, Carlsbad, CA). Following it, Reverse Transcription Kit (Thermo Scientific, Waltham, MA, USA) was adopted for cDNA synthesis. Next, SYBR-Green Real-Time PCR Kit (Qiagen, Zeeland, Netherlands) was exploited to carry out RT-qPCR. In the end, relative gene expression was computed by 2−∆∆Ct method. The normalized control was GAPDH or U6. The primer sequences were provided in Table 1 .
The sequences of primers used in RT-qPCR
| Primers | Sequences (5′–3′) |
|---|---|
| DARS-AS1-F | CATCGGGACACGGAACTGG |
| DARS-AS1-R | TGCAAAGAACTGCAGAAGACAC |
| miR-628-5p-F | GCCGAGATGCTGACATATTTAC |
| miR-628-5p-R | CTCAACTGGTGTCGTGGA |
| MGEA5-F | TGTGGCCAAAAGCATGATGG |
| MGEA5-R | GTACAAGAAAGTTGGGCACAGG |
| CACNB2-F | GAAGGCAATCATAGGCGAGC |
| CACNB2-R | GACCCTGCAGTACTCTGCTT |
| JAG1-F | CAAACACCAGCAGAAAGCCC |
| JAG1-R | TAAGTCAGCAACGGCCTCAG |
| ARRDC3-F | GAGACCACTGAGACGAGCG |
| ARRDC3-R | TTCACCTTTCCCAGCACCAT |
| VCAN-F | CTCGCAGAAACTGCATCACC |
| VCAN-R | CTGAGGTTGGACTGTGGCTT |
| GAPDH-F | CTCTGCTCCTCCTGTTCGAC |
| GAPDH-R | TTCCCGTTCTCAGCCTTGAC |
| U6-F | TCATCAGAAACAGTGGAGGT |
| U6-R | CATCCTTACACAGGAGCCAT |
6
Quantitative Analysis of lncRNA and miRNA Expression
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Total RNA was extracted from tumor tissues and cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was generated with a reverse transcription kit from Takara (Dalian, China). Amplification reactions included 2 µl of Mix, 2 µl of RNA and 6 µl of ddH2O. The thermal cycling protocol was as follows: 37 °C for 15 min, 85 °C for 5 s and 4 °C for 15 min. The expression of SNHG16 and SLC2A4 was detected by using a SYBR Green Real-Time PCR Kit (Qiagen, Germany). miR-302b-3p expression was analyzed with a Hairpin-it™ miRNA qPCR kit (GenePharma, China). Amplification of the transcripts involved an initial denaturation step at 95 °C for 2 min followed by 40 cycles at 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. The mRNA and miRNA expression data are presented as fold changes in the mRNA/miRNA abundance normalized to β-actin or U6 snRNA expression. The primer sequences used in this study are presented in Table 1 .
Primer sequences used for qRT-PCR
| Gene | Primer sequences |
|---|---|
| SNHG16 | |
| F | 5ʹ-CAGAATGCCATGGTTTCCCC-3ʹ′ |
| R | 5ʹ-TGGCAAGAGACTTCCTGAGG-3ʹ |
| miR-302b-3p | |
| Loop | 5′-GTCGTATCCAGTCCAGGGACCGAGGACTGGATACGACCTACTA-3′ |
| F | 5′-GCGTAAGTGCTTCCATGTT-3′ |
| R | 5′-TCCAGGGACCGAGGA-3′ |
| SLC2A4 | |
| F | 5′-TGGCTGGGTTCTCCAACTG-3′ |
| R | 5′-CTGGAAACCCATGCCAATG-3′ |
| β-actin | |
| F | 5′-ACCGAGCGCGGCTACAG-3′ |
| R | 5′-CTTAATGTCACGCACGATTTCC-3′ |
| U6 snRNA | |
| F | 5′ -CTCGCTTCGGCAGCACA-3′ |
| R | 5′-AACGCTTCACGAATTTGCGT-3′ |
7
Quantitative Analysis of SNHG16, SLC2A4 and miR-302b-3p Expression
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Total RNA was extracted from tumor tissues and cells by TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. cDNAs are produced by the reverse transcript kit from Takara (Dalian, China). SNHG16 and SLC2A4 expression was detected by using SYBR Green real-time PCR Kit (Qiagen, Germany). miR-302b-3p expression was analyzed by Hairpin-it TM miRNAs qPCR kit (GenePharma, China). The data of mRNA and miRNA are presented as the fold change in mRNA/miRNA abundance normalized against the β-actin or U6 snRNA. The primer sequences used in this study are presented in Table 1.
Table 1 Primer sequences for qRT-PCR
Table 1 Primer sequences for qRT-PCR
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