Patient tumor tissue samples were analyzed using qRT-PCR to explore the relationship between imaging features and gene expression. TRIzol reagent (Invitrogen) kits were used to extract total RNAs from the samples. According to the manufacturer's instructions, stem-loop antisense primer mix and AMV transcriptase (TaKaRa, China) kits were used to reverse transcribe these total RNAs into cDNAs. Finally, qRT-PCR was performed in the ABI 7500 real-time PCR system (Applied Biosystems, CA, USA) using an SYBR QPCR kit (Toyobo, Osaka, Japan). The primer sequences used are as follows: for ADRB1, forward: 5′-TCTACGTGCCCCTGTGCAT-3′ and reverse: 5′-TCGATCTTCTTCACCTGCTTCTG-3′; for GAPDH, forward: 5′-AAGAAGGTGGTGAAGCAGGC-3′ and reverse: 5′-TCCACCACCCAGTTGCTGTA-3′; for GAPDH, forward: 5′-AAGAAGGTGGTGAAGCAGGC-3′ and reverse: 5′-TCCACCACCCAGTTGCTGTA-3′ (47 (link)). GAPDH was used as a reference to normalize the same gene expressions between samples. The 2−ΔΔCt method was used to calculate the relative expression of the target gene. Gene expression was further normalized between samples before subsequent analysis.
Sybr qpcr kit
Manufactured by Toyobo
Sourced in Japan
The SYBR QPCR kit is a reagent system used for quantitative polymerase chain reaction (qPCR) analysis. It contains all the necessary components, including a DNA-binding fluorescent dye, to perform real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (6 mentions)
Stem loop antisense primer mix, by Takara Bio (2 mentions)
Amv transcriptase, by Takara Bio (2 mentions)
Mirneasy isolation kit, by Qiagen (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Tiangen Biotech (1 mentions)
Avian myeloblastosis virus transcriptase, by Takara Bio (1 mentions)
Mirneasy plasma kit, by Qiagen (1 mentions)
Rt pcr kit, by Takara Bio (1 mentions)
Lightcycler 480 thermal cycler, by Roche (1 mentions)
8 protocols using sybr qpcr kit
1
Exploring Tumor Gene Expression via qRT-PCR
2
miR-30e and NLRP3 Quantification Protocol
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For miR-30e quantitative analysis, total RNA was isolated using the miRNeasy isolation kit (Qiagen); 2 μg of retrieved total RNA was reversely transcribed using stem-loop antisense primer mix and AMV transcriptase (TaKaRa, China) according to the manufacturer's instructions. Extraction of total RNA of RAW 264.7 cells using a TRIzol reagent (Invitrogen) was performed as described. Quantitative assay of NLRP3 expressions was performed using a SYBR QPCR kit (Toyobo, Osaka, Japan). Real-time PCR was routinely performed on an ABI 7500 real-time PCR system (Applied Biosystems, CA, USA). The primer sequences were as follows: for miR-30e, sense: 5′-ACACTCCAGCTGGGTGTAAACATCCTTGAC-3′ and antisense: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTTCCA-3′; for U6, sense: 5′-CTCGCTTCGGCAGCACA-3′ and antisense: 5′-A ACGCTTCACGAATTTGCGT-3′. Finally, the relative amounts of miR-30e to U6 and NLRP3 to GAPDH were calculated using the 2−∆∆Ct method.
3
Quantitative Gene Expression Analysis
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Total RNA from SNpc tissues or BV-2 cells were extracted using RNAiso Plus Reagent (Takara, Dalian, China) and reverse transcription was performed with the SuperScriptIII First-Strand Synthesis system. Quantitative assay of genes expressions was performed using a SYBR QPCR Kit (Toyobo, Osaka, Japan) and ABI 7500 real-time PCR system (Applied Biosystems, CA, USA). The gene expression was normalized to the GAPDH and calculated using the ΔCT method. The specific primer sequences used were listed in Table S1.
4
Quantifying PGC-1α mRNA Expression
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The samples were homogenized in in TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturer’s instructions. 1 μg of RNA was reverse-transcribed to cDNA according to the manufacturer’s instructions (Thermo Scientific, San Jose, CA). A SYBR QPCR Kit (Toyobo, Osaka, Japan) was used in associated with ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) to detect PGC-1α mRNA expression. Mouse GAPDH was used as an endogenous control. The primer sequences were as follows: PGC-1α, 5′-TACGCAGGTCGAACGAAACT-3′ and 5′-GAAGGGGTCGCCCTTGTTC-3′; GAPDH, 5′-GGGCACGAAGGCTCATCATT-3′ and 5′-AGAAGGCTGGGGCTCATTTG-3′.
5
Quantitative Analysis of miRNA-223-3p
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Total RNA was extracted from DCs using TRIzol reagent (Invitrogen) according to the manufacturer's protocol and was reversely transcripted to cDNA with specific stem-loop primers by using a reverse transcription kit (Tiangen Biotech Co., Ltd., China). Quantitative PCR analysis of miRNAs was performed using an SYBR qPCR kit (Toyobo, Japan) and the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). The relative expression level of miRNAs was normalized to the internal control (U6) using the 2-ΔΔCt cycle threshold method. The primers for miR-223-3p and U6 were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China).
6
Quantifying ClC-5 Expression in Osteosarcoma
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The osteosarcoma samples were homogenized in TRIzol reagent (Invitrogen, CA, USA) and total RNA was isolated according to the manufacturer’s instructions. One microgram of RNA was reverse-transcribed to cDNA according to the manufacturer’s instructions (Thermo Fisher Scientific Inc., IL, USA). A SYBR QPCR Kit (Toyobo, Osaka, Japan) was used in associated with ABI 7500 real-time PCR system (Applied Biosystems, CA, USA) to detect ClC-5 mRNA expression. Human 18S rRNA was used as an endogenous control. The primer sequences were as follows: ClC-5, 5’-GTGAGGGAGAAATCCAGA-3’ and 5’-TTGATGATCAGCGTCCA-3’; 18S rRNA, 5’-CGGCTACCACATCCAAGGAA-3’ and 5’-CTGGAATTACCGCGGCT-3’.
7
Quantitative Analysis of miR-30e and Related Genes
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TRIZOL reagent (Invitrogen; Carlsbad, CA, USA) was used to extract total ribonucleic acids (RNAs). For the quantitative analysis of miR-30e, total RNA was isolated using the miRNeasy plasma kit (QIAGEN; Hilden, Germany). According to the manufacturer’s instructions, the total RNA was extracted using stem-loop antisense primer mix and avian myeloblastosis virus transcriptase (TaKaRa; Dalian, China) reverse transcription complementary deoxyribonucleic acid. The SYBR QPCR kit (Toyobo, Osaka, Japan) was then used to perform qRT-PCR on an ABI 7500 real-time PCR system (Applied Biosystems; CA, USA) (17 (link)). The primer-pair sequences used were: TUG1, 5'-CTGAAGAAAGGCAACATC-3' (forward) and 5'-GTAGGCTACTACAGGATTTG-3' (reverse); miR-30e, 5'-ACACTCCAGCTGGGT GTAAACATCCTTGAC-3' (forward) and 5'-CTCAACTGGTGTCGTGGAGTCGG CAATTCAGTTGAGCTTCCA-3' (reverse); NPPB, 5'-CTTTCCTGGGAGGTCGT TCC-3' (forward) and 5'-GTTGCGCTGCTCCTGTAAC-3' (reverse); U6, 5'-CTCGCT TCGGCAGCACA-3' (forward) and 5'-AACGCTTCACGAATTTGCGT-3' (reverse); and GAPDH, 5'-AAGAAGGTGGTGAAGCAGGC-3' (forward) and reverse, 5'-TCCACCACCCAGTTGCTGTA-3' (reverse). The quantification of messenger RNA (mRNA) and miRNA was performed using the 2−ΔΔCt method and was normalized by glyceraldehyde-3-phosphate dehydrogenase or U6.
8
POFUT1 Gene Expression Quantification
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POFUT1 messenger RNA (mRNA) expression was detected in GC cells using quantitative real-time polymerase chain reaction (q-PCR). Cells were treated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for RNA extraction. Complementary DNA (cDNA) was synthesized using an RT-PCR kit (TaKaRa, Tokyo, Japan) and amplified using q-PCR, according to the protocol of the SYBR q-PCR Kit (Toyobo, Osaka, Japan) using a Roche LightCycler 480 thermal cycler (Roche, Basel, Switzerland). The primers used were as follows: POFUT1: 5′-AACCAGGCCGATCACTTCTTG-3′ (forward); 5′-GTTGGTGAAAGGAGGCTTGTG-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5′-GGAGCGAGATCCCTCCAAAAT-3′ (forward); 5′-GGCTGTTGTCATACTTCTCATGG-3′ (reverse). Gene transcripts were normalized to GAPDH and the relative fold change was calculated using the 2 -ΔΔCT method.
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